Stable intrachromosomal biomarkers of past exposure to densely ionizing radiation in several chromosomes of exposed individuals

A multicolor banding (mBAND) fluorescence in situ hybridization technique was used to investigate the presence inhuman populations of a stable biomarker-intrachromosomal chromosome aberrations-of past exposure to high-LET radiation. Peripheral blood lymphocytes were taken from healthy Russian nuclear workers occupationally exposed from 1949 onward to either plutonium, gamma rays or both. Metaphase spreads were produced and chromosomes 1 and 2 were hybridized with mBAND FISH probes and scored for intra-chromosomal aberrations. A large yield of intrachromosomal aberrations was observed in both chromosomes of the individuals exposed to high doses of plutonium, whereas there was no significant increase over the (low) background control rate in the population who were exposed to high doses of gamma rays.Interchromosome aberration yields were similar in both the high plutonium and the high gamma-ray groups. These results for chromosome 1 and 2 confirm and extend data published previously for chromosome 5. Intrachromosomal aberrations thus represent a potential biomarker for past exposure to high-LET radiations such as alpha particles and neutrons and could possibly be used as a biodosimeter to estimate both the dose and type of radiation exposure in previously exposed populations.     

mBAND analysis for high- and low-LET radiation-induced chromosome aberrations: a review

During long-term space travel or cancer therapy, humans are exposed to high linear energy transfer (LET) energetic heavy ions. High-LET radiation is much more effective than low-LET radiation in causing various biological effects, including cell inactivation, genetic mutations, cataracts and cancer induction. Most of these biological endpoints are closely related to chromosomal damage, and cytogenetic damage can be utilized as a biomarker for radiation insults. Epidemiological data, mainly from survivors of the atomic bomb detonations in Japan, have enabled risk estimation from low-LET radiation exposures. The identification of a cytogenetic signature that distinguishes high- from low-LET exposure remains a long-term goal in radiobiology. Recently developed fluorescence in situ hybridization (FISH)-painting methodologies have revealed unique endpoints related to radiation quality. Heavy-ions induce a high fraction of complex-type exchanges, and possibly unique chromosome rearrangements. This review will concentrate on recent data obtained with multicolor banding in situ hybridization (mBAND) methods in mammalian cells exposed to low- and high-LET radiations. Chromosome analysis with mBAND technique allows detection of both inter- and intrachromosomal exchanges, and also distribution of the breakpoints of aberrations.     

Association of inter- and intrachromosomal exchanges with the distribution of low- and high-LET radiation-induced breaks in chromosomes

To study the effects of low- and high-linear energy transfer (LET) radiation on break locations within a chromosome, we exposed human epithelial cells in vitro to (137)Cs γ rays at both low and high dose rates, secondary neutrons at a low dose rate, and 600 MeV/u iron ions at a high dose rate. Breakpoints were identified using multicolor banding in situ hybridization (mBAND), which paints chromosome 3 in 23 different colored bands. For all four radiation scenarios, breakpoint distributions were found to be different from the predicted distribution based on band width. Detailed analysis of chromosome fragment ends involved in inter- or intrachromosomal exchanges revealed that the distributions of fragment ends participating in interchromosomal exchanges were similar between the two low-LET radiation dose rates and between the two high-LET radiation types, but the distributions were less similar between low- and high-LET radiations. For fragment ends participating in intrachromosomal exchanges, the distributions for all four radiation scenarios were similar, with clusters of breaks found in three regions. Analysis of the locations of the two fragment ends in chromosome 3 that joined to form an intrachromosomal exchange demonstrated that two breaks with a greater genomic separation can be more likely to rejoin than two closer breaks, indicating that chromatin folding can play an important role in the rejoining of chromosome breaks. Comparison of the breakpoint distributions to the distributions of genes indicated that the gene-rich regions do not necessarily contain more breaks. In general, breakpoint distributions depend on whether a chromosome fragment joins with another fragment in the same chromosome or with a fragment from a different chromosome.     

Repair of DNA damage induced by accelerated heavy ions in mammalian cells proficient and deficient in the non-homologous end-joining pathway

Human and rodent cells proficient and deficient in non-homologous end joining (NHEJ) were irradiated with X rays, 70 keV/microm carbon ions, and 200 keV/microm iron ions, and the biological effects on these cells were compared. For wild-type CHO and normal human fibroblast (HFL III) cells, exposure to iron ions yielded the lowest cell survival, followed by carbon ions and then X rays. NHEJ-deficient xrs6 (a Ku80 mutant of CHO) and 180BR human fibroblast (DNA ligase IV mutant) cells showed similar cell survival for X and carbon-ion irradiation (RBE = approximately 1.0). This phenotype is likely to result from a defective NHEJ protein because xrs6-hamKu80 cells (xrs6 cells corrected with the wild-type KU80 gene) exhibited the wild-type response. At doses higher than 1 Gy, NHEJ-defective cells showed a lower level of survival with iron ions than with carbon ions or X rays, possibly due to inactivation of a radioresistant subpopulation. The G(1) premature chromosome condensation (PCC) assay with HFL III cells revealed LET-dependent impairment of repair of chromosome breaks. Additionally, iron-ion radiation induced non-repairable chromosome breaks not observed with carbon ions or X rays. PCC studies with 180BR cells indicated that the repair kinetics after exposure to carbon and iron ions behaved similarly for the first 6 h, but after 24 h the curve for carbon ions approached that for X rays, while the curve for iron ions remained high. These chromosome data reflect the existence of a slow NHEJ repair phase and severe biological damage induced by iron ions. The auto-phosphorylation of DNA-dependent protein kinase catalytic subunits (DNA-PKcs), an essential NHEJ step, was delayed significantly by high-LET carbon- and iron-ion radiation compared to X rays. This delay was further emphasized in NHEJ-defective 180BR cells. Our results indicate that high-LET radiation induces complex DNA damage that is not easily repaired or is not repaired by NHEJ even at low radiation doses such as 2 Gy.     

Relative effectiveness of HZE iron-56 particles for the induction of cytogenetic damage in vivo

One of the risks of prolonged manned space flight is the exposure of astronauts to radiation from galactic cosmic rays, which contain heavy ions such as (56)Fe. To study the effects of such exposures, experiments were conducted at the Brookhaven National Laboratory by exposing Wistar rats to high-mass, high-Z, high-energy (HZE) particles using the Alternating Gradient Synchrotron (AGS). The biological effectiveness of (56)Fe ions (1000 MeV/nucleon) relative to low-LET gamma rays and high-LET alpha particles for the induction of chromosome damage and micronuclei was determined. The mitotic index and the frequency of chromosome aberrations were evaluated in bone marrow cells, and the frequency of micronuclei was measured in cells isolated from the trachea and the deep lung. A marked delay in the entry of cells into mitosis was induced in the bone marrow cells that decreased as a function of time after the exposure. The frequencies of chromatid aberrations and micronuclei increased as linear functions of dose. The frequency of chromosome aberrations induced by HZE particles was about 3.2 times higher than that observed after exposure to (60)Co gamma rays. The frequency of micronuclei in rat lung fibroblasts, lung epithelial cells, and tracheal epithelial cells increased linearly, with slopes of 7 x 10(-4), 12 x 10(-4), and 11 x 10(-4) micronuclei/binucleated cell cGy(-1), respectively. When genetic damage induced by radiation from (56)Fe ions was compared to that from exposure to (60)Co gamma rays, (56)Fe-ion radiation was between 0.9 and 3.3 times more effective than (60)Co gamma rays. However, the HZE-particle exposures were only 10-20% as effective as radon in producing micronuclei in either deep lung or tracheal epithelial cells. Using microdosimetric techniques, we estimated that 32 cells were hit by delta rays for each cell that was traversed by the primary HZE (56)Fe particle. These calculations and the observed low relative effectiveness of the exposure to HZE particles suggest that at least part of the cytogenetic damage measured was caused by the delta rays. Much of the energy deposited by the primary HZE particles may result in cell killing and may therefore be "wasted" as far as production of detectable micronuclei is concerned. The role of wasted energy in studies of cancer induction may be important in risk estimates for exposure to HZE particles.     

Chromosome intrachanges and interchanges detected by multicolor banding in lymphocytes: searching for clastogen signatures in the human genome

Genomic fingerprints of mutagenic agents would have wide applications in the field of cancer biology, epidemiology and prevention. The differential spectra of chromosomal aberrations induced by different clastogens suggest that ratios of specific aberrations can be exploited as biomarkers of carcinogen exposure. We have tested this hypothesis using the novel technique of multicolor banding in situ hybridization (mBAND) in human peripheral blood lymphocytes exposed in vitro to X rays, neutrons, heavy ions, or the restriction endonuclease AluI. In the heavy-ion-irradiated cells, we further analyzed aberrations in chromosome 5 using multicolor FISH (mFISH). Contrary to the expectations of biophysical models, our results do not support the use of the ratios of inter-/intrachromosomal exchanges or intra-/interarm intrachanges as fingerprints of exposure to densely ionizing radiation. However, our data point to measurable differences in the ratio of complex/simple interchanges after exposure to different clastogens. These data should be considered in current biophysical models of radiation action in living cells.     

Karyotypes of human lymphocytes exposed to high-energy iron ions

Chromosomal aberrations were analyzed using multicolor fluorescence in situ hybridization (mFISH) in human peripheral blood lymphocytes after in vitro exposure to gamma rays or accelerated (56)Fe ions (1 GeV/nucleon, 145 keV/microm) at Brookhaven National Laboratory (Upton, NY). Doses of 0.3 and 3 Gy were used for both radiation types. Chromosomes were prematurely condensed by a phosphatase inhibitor (calyculin A) to avoid the population selection bias observed at metaphase as a result of the severe cell cycle delays induced by heavy ions. A total of 1053 karyotypes (G(2) and M phases) were analyzed in irradiated lymphocytes. Results revealed different distribution patterns for chromosomal aberrations after low- and high-LET radiation exposures: Heavy ions induced a much higher fraction of cells with multiple aberrations, while the majority of the aberrant cells induced by low doses of gamma rays contained a single aberration. The high fraction of complex-type exchanges after heavy ions leads to an overestimation of simple-type asymmetrical interchanges (dicentrics) from analysis of Giemsa-stained samples. However, even after a dose of 3 Gy iron ions, about 30% of the cells presented no complex-type exchanges. The involvement of individual chromosomes in exchanges was similar for densely and sparsely ionizing radiation, and no statistically significant evidence of a nonrandom involvement of specific chromosomes was detected.     

Chromosomes lacking telomeres are present in the progeny of human lymphocytes exposed to heavy ions

High-charge and energy (HZE) nuclei represent one of the main health risks for human space exploration, yet little is known about the mechanisms responsible for the high biological effectiveness of these particles. We have used in situ hybridization probes for cross-species multicolor banding (RxFISH) in combination with telomere detection to compare yields of different types of chromosomal aberrations in the progeny of human peripheral blood lymphocytes exposed to either high-energy iron ions or gamma rays. Terminal deletions showed the greatest relative variation, with many more of these types of aberrations induced after exposure to accelerated iron ions (energy 1 GeV/nucleon) compared with the same dose of gamma rays. We found that truncated chromosomes without telomeres could be transmitted for at least three cell cycles after exposure and represented about 10% of all aberrations observed in the progeny of cells exposed to iron ions. On the other hand, the fraction of cells carrying stable, transmissible chromosomal aberrations was similar in the progeny of cells exposed to the same dose of densely or sparsely ionizing radiation. The results demonstrate that unrejoined chromosome breaks are an important component of aberration spectra produced by the exposure to HZE nuclei. This finding may well be related to the ability of such energetic particles to produce untoward late effects in irradiated organisms.     

High yields of isochromatid breaks and successive formation of chromosome exchanges may lead to reproductive cell death following high-LET irradiation

To clarify the relationship between cell death and chromosomal aberrations following exposure to heavy-charged ion particles beams, exponentially growing Human Salivary Gland Tumor cells (HSG cells) were irradiated with various kinds of high energy heavy ions; 13 keV/μm carbon ions as a low-LET charged particle radiation source, 120 keV/μm carbon ions and 440 keV/μm iron ions as high-LET charged particle radiation sources. X-rays (200 kVp) were used as a reference. Reproductive cell death was evaluated by clonogenic assays, and the chromatid aberrations in G2/M phase and their repairing kinetics were analyzed by the calyculin A induced premature chromosome condensation (PCC) method. High-LET heavy-ion beams introduced much more severe and un-repairable chromatid breaks and isochromatid breaks in HSG cells than low-LET irradiation. In addition, the continuous increase of exchange aberrations after irradiation occurred in the high-LET irradiated cells. The cell death, initial production of isochromatid breaks and subsequent formation of chromosome exchange seemed to be depend similarly on LET with a maximum RBE peak around 100–200 keV/μm of LET value. Conversely, un-rejoined isochromatid breaks or chromatid breaks/gaps seemed to be less effective in reproductive cell death. These results suggest that the continuous yield of chromosome exchange aberrations induced by high-LET ionizing particles is a possible reason for the high RBE for cell death following high-LET irradiation, alongside other chromosomal aberrations additively or synergistically.     

Association between G2-phase block and repair of radiation-induced chromosome fragments in human lymphocytes.

We have studied the induction of chromosomal aberrations in human lymphocytes exposed in G0 to X rays or carbon ions. Aberrations were analyzed in G0, G1, G2 or M phase. Analysis during the interphase was performed by chemically induced premature chromosome condensation, which allows scoring of aberrations in G1, G2 and M phase; fusion-induced premature chromosome condensation was used to analyze the damage in G0 cells after incubation for repair; M-phase cells were obtained by conventional Colcemid block. Aberrations were scored by Giemsa staining or fluorescence in situ hybridization (chromosomes 2 and 4). Similar yields of fragments were observed in G1 and G2 phase, but lower yields were scored in metaphase. The frequency of chromosomal exchanges was similar in G0 (after repair), G2 and M phase for cells exposed to X rays, while a lower frequency of exchanges was observed in M phase when lymphocytes were irradiated with high-LET carbon ions. The results suggest that radiation-induced G2-phase block is associated with unrejoined chromosome fragments induced by radiation exposure during G0.     

Delayed cell cycle progression in human lymphoblastoid cells after exposure to high-LET radiation correlates with extremely localized DNA damage

To compare the genotoxic effects of high-LET ionizing radiation to those of low-LET radiation, we investigated the responses of human lymphoblastoid cells to DNA damage TK6 after treatment with either low-LET X rays or high-LET iron ions (1000 keV/microm). A highly localized distribution of gammaH2AX/RAD51 foci was observed in the nuclei of cells irradiated with iron ions, in sharp contrast to cells exposed to X rays, where the distribution of foci was much more uniform. This implied the occurrence of a relatively high frequency of closely spaced double-strand breaks, i.e. clustered DNA damage, after iron-ion exposure. Despite the well-established notion that clustered DNA damage is refractory to repair compared to isolated DNA lesions, there were no significant differences in the levels of clonogenic survival and apoptosis between cells treated with iron ions or X rays. Strikingly, however, cells accumulated in G(2)/M phase to a much lesser extent after iron-ion exposure than after X-ray exposure. This differential accumulation could be attributed to a much slower evacuation of the S-phase compartment in the case of cells irradiated with iron ions. Taken together, our results indicate that, relative to the situation for low-LET X rays, exposure to high-LET iron ions results in a substantially greater inhibition of S-phase progression as a result of a higher frequency of DNA replication-blocking clustered DNA damage.     

Transmissible and nontransmissible complex chromosome aberrations characterized by three-color and mFISH define a biomarker of exposure to high-LET alpha particles

Insertions have been proposed as potential stable biomarkers of chronic high-LET radiation exposure. To examine this in vitro, we irradiated human peripheral blood lymphocytes in G(0) with either 50 cGy (238)Pu alpha particles (LET 121.4 keV/microm) or 3 Gy 250 kV X rays and stimulated their long-term culture up to approximately 22 population doublings postirradiation. Mitotic cells were harvested at regular intervals throughout this culture period and were assayed for chromosome aberrations using the techniques of three-color and 24-color mFISH. We observed the stable persistence of transmissible-type complex rearrangements, all involving at least one insertion. This supports the hypothesis that insertions are relevant indicators of exposure to high-LET radiation. However, one practical caveat of insertions being effective biomarkers is that their frequency is low due to the complexity and cell lethality of the majority of alpha-particle-induced complexes. Therefore, we propose a "profile of damage" that relies on the presence of insertions, a low frequency of stable simple reciprocal translocations (2B), and, significantly, the complexity of the damage initially induced. We suggest that the complexity of first- and second-division alpha-particle-induced nontransmissible complex aberrations reflects the structure of the alpha-particle track and as a consequence adds radiation-quality specificity to the biomarker, increasing the signal:noise ratio of the characteristic 2B:insertion ratio.     

Initial yields of DNA double-strand breaks and DNA Fragmentation patterns depend on linear energy transfer in tobacco BY-2 protoplasts irradiated with helium, carbon and neon ions

The ability of ion beams to kill or mutate plant cells is known to depend on the linear energy transfer (LET) of the ions, although the mechanism of damage is poorly understood. In this study, DNA double-strand breaks (DSBs) were quantified by a DNA fragment-size analysis in tobacco protoplasts irradiated with high-LET ions. Tobacco BY-2 protoplasts, as a model of single plant cells, were irradiated with helium, carbon and neon ions having different LETs and with gamma rays. After irradiation, DNA fragments were separated into sizes between 1600 and 6.6 kbp by pulsed-field gel electrophoresis. Information on DNA fragmentation was obtained by staining the gels with SYBR Green I. Initial DSB yields were found to depend on LET, and the highest relative biological effectiveness (about 1.6) was obtained at 124 and 241 keV/microm carbon ions. High-LET carbon and neon ions induced short DNA fragments more efficiently than gamma rays. These results partially explain the large biological effects caused by high-LET ions in plants.     

Persistence of chromosome aberrations in mice acutely exposed to 56Fe+26 ions

Space exploration has the potential to yield exciting and significant discoveries, but it also brings with it many risks for flight crews. Among the less well studied of these are health effects from space radiation, which includes the highly charged, energetic particles of elements with high atomic numbers that constitute the galactic cosmic rays. In this study, we demonstrated that 1 Gy iron ions acutely administered to mice in vivo resulted in highly complex chromosome damage. We found that all types of aberrations, including dicentrics as well as translocations, insertions and acentric fragments, disappear rapidly with time after exposure, probably as a result of the death of heavily damaged cells, i.e. cells with multiple and/or complex aberrations. In addition, numerous cells have apparently simple exchanges as their only aberrations, and these cells appear to survive longer than heavily damaged cells. Eight weeks after exposure, the frequency of cells showing cytogenetic damage was reduced to less than 20% of the levels evident at 1 week, with little further decline apparent over an additional 8 weeks. These results indicate that exposure to 1 Gy iron ions produces heavily damaged cells, a small fraction of which appear to be capable of surviving for relatively long periods. The health effects of exposure to high-LET radiation in humans on prolonged space flights should remain a matter of concern.     

Truly incomplete and complex exchanges in prematurely condensed chromosomes of human fibroblasts exposed in vitro to energetic heavy ions

Confluent human fibroblast cells (AG1522) were irradiated with gamma rays, 490 MeV/nucleon silicon ions, or iron ions at either 200 or 500 MeV/nucleon. The cells were allowed to repair at 37 degrees C for 24 h after exposure, and a chemically induced premature chromosome condensation (PCC) technique was used to condense chromosomes in the G2 phase of the cell cycle. Incomplete and complex exchanges were analyzed in the irradiated samples. To verify that chromosomal breaks were truly unrejoined, chromosome aberrations were analyzed using a combination of whole-chromosome specific probes and probes specific for the telomere region of the chromosome. Results showed that the frequency of unrejoined chromosome breaks was higher after irradiation with the heavy ions of high LET, and consequently the ratio of incomplete to complete exchanges increased steadily with LET up to 440 keV/microm, the highest LET included in the present study. For samples exposed to 200 MeV/nucleon iron ions, chromosome aberrations were analyzed using the multicolor FISH (mFISH) technique, which allows identification of both complex and truly incomplete exchanges. Results of the mFISH study showed that 0.7 and 3 Gy iron ions produced similar ratios of complex to simple exchanges and incomplete to complete exchanges; these ratios were higher than those obtained after exposure to 6 Gy gamma rays. After 0.7 Gy of iron ions, most complex aberrations were found to involve three or four chromosomes, which is a likely indication of the maximum number of chromosome domains traversed by a single iron-ion track.     

Repair of HZE-particle-induced DNA double-strand breaks in normal human fibroblasts

DNA damage generated by high-energy and high-Z (HZE) particles is more skewed toward multiply damaged sites or clustered DNA damage than damage induced by low-linear energy transfer (LET) X and gamma rays. Clustered DNA damage includes abasic sites, base damages and single- (SSBs) and double-strand breaks (DSBs). This complex DNA damage is difficult to repair and may require coordinated recruitment of multiple DNA repair factors. As a consequence of the production of irreparable clustered lesions, a greater biological effectiveness is observed for HZE-particle radiation than for low-LET radiation. To understand how the inability of cells to rejoin DSBs contributes to the greater biological effectiveness of HZE particles, the kinetics of DSB rejoining and cell survival after exposure of normal human skin fibroblasts to a spectrum of HZE particles was examined. Using gamma-H2AX as a surrogate marker for DSB formation and rejoining, the ability of cells to rejoin DSBs was found to decrease with increasing Z; specifically, iron-ion-induced DSBs were repaired at a rate similar to those induced by silicon ions, oxygen ions and gamma radiation, but a larger fraction of iron-ion-induced damage was irreparable. Furthermore, both DNA-PKcs (DSB repair factor) and 53BP1 (DSB sensing protein) co-localized with gamma-H2AX along the track of dense ionization produced by iron and silicon ions and their focus dissolution kinetics was similar to that of gamma-H2AX. Spatial co-localization analysis showed that unlike gamma-H2AX and 53BP1, phosphorylated DNA-PKcs was localized only at very specific regions, presumably representing the sites of DSBs within the tracks. Examination of cell survival by clonogenic assay indicated that cell killing was greater for iron ions than for silicon and oxygen ions and gamma rays. Collectively, these data demonstrate that the inability of cells to rejoin DSBs within clustered DNA lesions likely contributes to the greater biological effectiveness of HZE particles.     

Complex chromosomal rearrangements induced in vivo by heavy ions

It has been suggested that the ratio complex/simple exchanges can be used as a biomarker of exposure to high-LET radiation. We tested this hypothesis in vivo, by considering data from several studies that measured complex exchanges in peripheral blood from humans exposed to mixed fields of low- and high-LET radiation. In particular, we studied data from astronauts involved in long-term missions in low-Earth-orbit, and uterus cancer patients treated with accelerated carbon ions. Data from two studies of chromosomal aberrations in astronauts used blood samples obtained before and after space flight, and a third study used blood samples from patients before and after radiotherapy course. Similar methods were used in each study, where lymphocytes were stimulated to grow in vitro, and collected after incubation in either colcemid or calyculin A. Slides were painted with whole-chromosome DNA fluorescent probes (FISH), and complex and simple chromosome exchanges in the painted genome were classified separately. Complex-type exchanges were observed at low frequencies in control subjects, and in our test subjects before the treatment. No statistically significant increase in the yield of complex-type exchanges was induced by the space flight. Radiation therapy induced a high fraction of complex exchanges, but no significant differences could be detected between patients treated with accelerated carbon ions or X-rays. Complex chromosomal rearrangements do not represent a practical biomarker of radiation quality in our test subjects.     

Chromosome aberrations induced by dual exposure of protons and iron ions

During space travel, astronauts will be exposed to protons and heavy charged particles. Since the proton flux is high compared to HZE particles, on average, it is assumed that a cell will be hit by a proton before it is hit by an HZE ion. Although the effects of individual ion species on human cells have been investigated extensively, little is known about the effects of exposure to mixed beam irradiation. To address this, we exposed human epithelial cells to protons followed by HZE particles and analyzed chromosomal damage using the multicolor banding in situ hybridization (mBAND) procedure. With this technique, individually painted chromosomal bands on one chromosome allowed the identification of intra-chromosomal aberrations (inversions and deletions within a single painted chromosome) as well as inter-chromosomal aberrations (translocation to unpainted chromosomes). Our results indicated that chromosome aberration frequencies from exposures to protons followed by Fe ions did not simply decrease as the interval between the two exposures increased, but peak when the interval was 30 min.     

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.