Significant large-scale chromosome territory movement occurs as a result of mitosis, but not during interphase.

PURPOSE: To investigate large-scale relative movement (position change) of interphase chromosome territories (ICT), as indicated by the separation of chromosome derivatives following exposure to ionizing radiation. MATERIALS AND METHODS: A simple experiment was conducted to investigate large-scale movement of ICT, using whole chromosome 18 painting to measure the frequency of fluorescent ICT in irradiated lymphocytes, cultured over 9 days (seven cell cycles). After chromosome 18 painting, derivative chromosome territory separation was indicated by the observation of three fluorescent ICT in aberrant cells compared with the presence of two fluorescent ICT in normal cells. The frequencies of interphase nuclei containing three fluorescent chromosome territories for unirradiated resting lymphocytes and for lymphocytes acutely irradiated with 2.25 or 5.5 Gy 137Cs gamma-rays were measured for each culture time point of 0, 1, 2, 3, 4, 7 and 9 days. RESULTS: No significant difference was observed in the frequency of three ICT between the average of the controls and irradiated cells cultured for 0, 1 and 2 days. However, irradiated cells cultured for >or=3 days showed a significant increase in the frequency of three fluorescent ICT over those cultured for

Relationship between radiation-induced aberrations in individual chromosomes and their DNA content: effects of interaction distance.

PURPOSE: To study the effect of the interaction distance on the frequency of inter- and intrachromosome exchanges in individual chromosomes with respect to their DNA content. Assumptions: Chromosome exchanges are formed by misrejoining of two DNA double-strand breaks (DSB) induced within an interaction distance, d. It is assumed that chromosomes in G(0)/G(1) phase of the cell cycle occupy a spherical domain in a cell nucleus, with no spatial overlap between individual chromosome domains. RESULTS: Formulae are derived for the probability of formation of inter-, as well as intra-, chromosome exchanges relating to the DNA content of the chromosome for a given interaction distance. For interaction distances <1 microm, the relative frequency of interchromosome exchanges predicted by the present model is similar to that by Cigarran et al. (1998) based on the assumption that the probability of interchromosome exchanges is proportional to the "surface area" of the chromosome territory. The "surface area" assumption is shown to be a limiting case of d-->0 in the present model. The present model also predicts that the probability of intrachromosome exchanges occurring in individual chromosomes is proportional to their DNA content with correction terms. CONCLUSION: When the interaction distance is small, the "surface area" distribution for chromosome participation in interchromosome exchanges has been expected. However, the present model shows that for the interaction distance as large as 1 microm, the predicted probability of interchromosome exchange formation is still close to the surface area distribution. Therefore, this distribution does not necessarily rule out the formation of complex chromosomal aberrations by long-range misrejoining of DSB.

Fluorescence in situ hybridization of metaphase chromosomes in suspension.

PURPOSE: To describe a new method for FISH analysis of metaphase chromosomes in suspension. MATERIALS AND METHODS: Metaphase chromosomes in suspension were isolated from a Chinese hamster human hybrid cell line, 314-2 (1) Y, and a human cell line, GM 130B. During suspension hybridization, specific chromosomes were labeled from these two cell lines using either biotin-labeled human genomic DNA, a directly labeled human pancentromere DNA probe or a chromosome #1 locus-specific probe. RESULTS: The method allows, for the first time, recovery of large numbers of isolated individual hybridized chromosomes with good morphology for both human x hamster hybrid and human cell lines. The results showed that 46-73% of the starting number of total chromosomes can be recovered after a FISH in suspension procedure. The well-preserved morphology of hybridized metaphase chromosomes allowed (1) rapid detection of individual human and hamster chromosome aberrations, (2) rapid counting of the painted human chromosomes and (3) fast, clear detection of chromosome region-specific probes. This method offers a new tool to assay chromosomes and DNA: it offers the possibility to develop new techniques for sorting chromosomes based on FISH signals, for early detection and screening of genetic diseases and for bulk measurement of both balanced or unbalanced chromosomal exchanges and rearrangements. CONCLUSION: The potential of the method described should facilitate fast, sensitive population monitoring, and increase sensitivity of the measurements in chromosome-based biodosimetry.

Biodosimetry using chromosomal translocations measured by FISH in a population chronically exposed to low dose-rate 60Co gamma-irradiation.

PURPOSE: To evaluate the cumulative gamma-radiation personal exposure by analysing lymphocyte chromosome translocations using FISH painting and to compare FISH-derived biodoses with those derived from retrospective physical dose reconstruction in residents receiving chronic low dose-rate gamma-irradiation while living in radio-contaminated buildings. MATERIALS AND METHODS: Chromosome translocation frequencies were evaluated by scoring 933 to 3077 metaphases under fluorescence microscope for each of the five male and four female exposed individuals after they had relocated from the radioactive environment for 34-82 months. FISH painting was conducted using kits of whole-chromosome probes for chromosomes 1, 2 and 4 in orange and 3, 5 and 6 in green and counter-stained with 4',6-diamidino-2-phenylindole (DAPI). The retrospective dose estimation termed Taiwan Cumulative Dose (TCD) was conducted by assessment using detailed information of historical exposure and the environmental radioactivity for each apartment during previous residency. RESULTS: A total of 20 244 well-prepared metaphases were scored. Biodoses were calculated from the translocation frequencies and physical doses were estimated from detail questionnaires for each individual. The translocation frequencies measured ranged from 2.2x10(-3) to 26.8x10(-3) translocations per cell and the dose equivalent from 52.2 to 992.2mSv. A good correlation was observed between the physical and biodoses. A plot of TCD against FISH-derived doses produced D(fish) =0.65 D(TCD), when fitted by a linear model, and D(fish) = 0.53 D(TCD)+ 1.26x10(-4 ) D(2)(TCD), when fitted with a linear-quadratic model. Given the scatter in the data and the extremely small quadratic dose contribution, neither model could be ruled out. CONCLUSION: Chromosome translocations provide a valid method of dose estimation in extremely protracted low dose-rate gamma-radiation exposure. Validation of the TCD method by FISH-measured translocations supports the use of TCD for epidemiological studies.

A study to verify a reported excess of chromosomal aberrations in blood lymphocytes of Namibian uranium miners.

This report describes a study to verify an earlier report of excess chromosomal damage in the blood lymphocytes of uranium miners. Coded blood samples from 10 miners and 10 controls were analyzed conventionally for unstable aberrations and by FISH for translocations. Conventional analysis, scoring 1000 metaphases per subject, showed no significant difference between miners and controls in the frequencies of chromosome- and chromatid-type aberrations. Investigators at two laboratories undertook FISH analyses, each scoring 4000 metaphases per subject. When the data from each laboratory were examined separately, one found slightly more translocations in the miners while the other found fewer. In neither case was the difference significant at the 95% level of confidence. Combining the data likewise showed no significant excess of damage in the miners. This applied to simple one- and two-way translocations and to cells with complex exchanges. There was no correlation between levels of translocations and total lifetime doses from occupational and/or background irradiation. A borderline significant excess of rogue cells was found in the miners. This may be a chance observation, as these rare, highly abnormal cells are considered to be unrelated to radiation exposure and are probably due to a virus. The overall conclusion is that the frequency of chromosomal damage in the miners did not exceed that in the controls. Therefore, the result of the earlier study was not confirmed.

A comparative study on potential cytogenetic fingerprints for radiation LET in human lymphocytes.

PURPOSE: To carry out a comparative study on potential cytogenetic fingerprints for radiation LET in human metaphase lymphocytes. MATERIALS AND METHOD: Human lymphocytes were irradiated in vitro with 3.0 Gy 60Co gamma-rays, 0.9 Gy 3H beta-rays or 0.2 Gy 2.7 Mev neutrons. Detailed chromosome aberrations were analysed by combined FISH with pan-telomere staining and specific whole-chromosome painting (1, 2 and 4). Total chromosome translocations and insertions were also analysed by multicolour whole-chromosome painting (chromosomes 1, 2 and 4 orange, chromosomes 3, 5 and 6 green). RESULTS: Among the six proposed radiation cytogenetic fingerprints, the ratio of total simple translocations to insertions (I-ratio), showed the largest difference between low-LET 60Co gamma-ray and high-LET neutron radiation. The ratios of complete exchanges to incomplete rejoinings [S(I)-ratio] and dicentrics to interstitial deletions (H-ratio), showed a similar significant difference between low- and high-LET radiation. The ratios of centric rings to interstitial deletion (G-ratio) showed a trend of LET-related difference, but the difference was not significant in this data set. The ratios of dicentrics to centric rings (F-ratio) and apparent complete exchanges to hidden complete exchanges [S(II)-ratio], showed no difference between low- and high-LET radiation. In the 1426 radiation-induced chromosome aberrations observed after 52 h culture, evidence for sister-chromatid fusion but not telomere addition was found. CONCLUSION: Pan-telomere staining plus specific whole chromosome painting allows simultaneous and objective detection of complete or incomplete chromosome exchanges and interstitial or terminal deletions in human peripheral lymphocytes. Of the six proposed cytogenetic ratios, the I-ratio is the most effective cytogenetic fingerprint for distinguishing low-LET from high-LET radiation in human metaphase human lymphocytes.

Combined FISH with pan-telomeric PNA and whole chromosome-specific DNA probes to detect complete and incomplete chromosomal exchanges in human lymphocytes.

PURPOSE: To combine FISH with pan-telomeric peptide nucleic acid (PNA) and whole chromosome-specific DNA probes to detect complete and incomplete chromosome exchanges in human lymphocytes. MATERIALS AND METHODS: Human lymphocytes were irradiated in vitro with 0.9 Gy low dose-rate (0.019 Gy/h) tritium beta-rays. Metaphase spreads were treated with RNase, fixed in 1:3 acetic acid:methanol, and then further treated with KCl, proteinase K and fixed in 4% paraformaldehyde. Slides were denatured, hybridized for 1.5 h with an FITC-labelled telomeric PNA probe, and rehybridized overnight with a spectrum-orange whole-chromosome probe specific for chromosomes 1, 2 and 4. Hybridized spreads were washed with 70% formamide/20 x SSC and counterstained with DAPI. RESULTS: All three pairs of labelled chromosomes together with 92 telomeres were readily visible after hybridization. The whole chromosomes 1, 2 and 4 were painted orange, and all telomeres were stained green. Unpainted chromosomes were counterstained blue. In the observed 680 chromosome aberrations induced by tritium beta-rays in human lymphocytes after 52 h of culture, no evidence of telomere addition was detected. Incomplete and hidden complete exchanges and terminal deletions were definitively discriminated. CONCLUSION: The simultaneous detection of telomeres and specific whole chromosomes allows for the first time accurate analysis of complete and incomplete chromosome exchanges involving painted chromosomes in human lymphocytes.

Theoretical and experimental tests of a chromosomal fingerprint for densely ionizing radiation based on F ratios calculated from stable and unstable chromosome aberrations.

In the present study, F ratios for both stable chromosome aberrations, i.e. ratios of translocations to pericentric inversions, and unstable aberrations, i.e. dicentrics and centric rings, were measured using fluorescence in situ hybridization. F ratios for stable aberrations measured after exposure to low (2.89 Gy 60Co gamma rays) and high-LET (0.25 Gy 56Fe ions; 1.25 Gy 56Fe ions; 3.0 Gy 12C ions) radiation were 6.5 +/- 1.5, 4.7 +/- 1.6, 9.3 +/- 2.5 and 10.4 +/- 3.0, respectively. F ratios for unstable aberrations measured after low (2.89 Gy 60Co gamma rays) and high-LET (0.25 Gy 56Fe ions; 3.0 Gy 12C ions) radiations were 6.5 +/- 1.6, 6.3 +/- 2.3 and 11.1 +/- 3.7, respectively. No significant difference between the F ratios for low- and high-LET radiation was found. Further tests on the models for calculation of the F ratio proposed by Brenner and Sachs (Radiat. Res. 140, 134-142, 1994) showed that the F ratio may not be straightforward as a practical fingerprint for densely ionizing radiation.

Background ionizing radiation plays a minor role in the production of chromosome translocations in a control population.

PURPOSE: To obtain a relationship between background chromosome translocation frequency and age with translocation frequency measured to a high statistical precision, and to identify the role of background ionizing radiation in the production of chromosome translocations in a control population. MATERIALS AND METHODS: Lymphocytes from 35 healthy control individuals (15 females and 20 males) were scored, using fluorescence in situ hybridization, for the presence of chromosomal translocations. Translocation frequencies were measured to a high statistical precision (s.d. 25% or less for each individual). These control subjects were of varying ages, ranging from 0 (cord blood) to 98 years. RESULTS: In a total of 521,492 metaphases (203,754 genome equivalent cells) scored, an average of 5,822 genome equivalent cells per individual, 764 translocations were observed in the 35 individuals. The translocation frequencies ranged from 0 (for cord blood) to 0.0167 (for a 98 year old) translocations per cell. The average age and translocation frequency was 50 years and 0.004 translocations per cell, respectively. The best fit of the relationship between translocations and age was: Y=7x10(-4)+6.9x10(-6)A+1.35x10(-6)A2, which does not obey the linear relationship expected from chronic background radiation alone. The curvilinear relationship observed clearly shows that other endogenous and exogenous clastogens or clastogenic events, in addition to radiation, serve to generate chromosome translocations in control populations. CONCLUSION: The background translocation frequency in control individuals follows a curvilinear relationship with age. No significant variation was observed between individuals of the same age. Clastogenic processes of normal aging and physiological factors in additional to ionizing radiation play a major role in the production of chromosome translocations in a control population. Background radiation, however, appears to play a minor role in chromosome translocation production in control individuals living near sea level.

Alpha coefficient of dose-response for chromosome translocations measured by FISH in human lymphocytes exposed to chronic 60Co gamma rays at body temperature.

PURPOSE: To measure the alpha coefficient, the initial slope of the translocation dose-response curve, for 60Co gamma-rays in human lymphocytes. MATERIALS AND METHODS: Human lymphocytes were exposed to 0, 0.32, 0.62 and 0.92 Gy of chronic 60Co gamma-rays under conditions that reduce the metabolic stress to the cells. Chromosome translocation frequencies were measured using fluorescence in situ hybridization with a whole-chromosome probe cocktail specific for chromosomes 1, 2, 4 (orange) and 3, 5, 6 (green). RESULTS: A total of 72,383 metaphases were analysed (33,429 in exposed cells) in two individuals. The shape of the dose-response curves for translocations was linear, and alpha coefficient was measured as 0.024 +/- 0.002 translocations per cell per Gy for the combined data for two 24 year old male donors. CONCLUSION: The alpha coefficients measured after chronic exposure were in good agreement with that reported in the literature for acute, low-dose exposure of human lymphocytes to 60Co gamma-rays.

Exposure temperature, but not donor age, is a confounding factor for in vitro translocation production by chronic irradiation.

PURPOSE: To assess the effects of incubation temperature during irradiation, and of donor age, on the in vitro induction of chromosomal translocations in human lymphocytes. MATERIAL AND METHODS: Lymphocytes from six human male donors were scored, using fluorescence in situ hybridization, for the presence of chromosomal translocations involving chromosomes 1 to 6 after in vitro, chronic exposure (delivered continuously over 48 h at 37 degrees C or at 20 degrees C) to tritium beta-rays or 60Co gamma-rays. RESULTS: No age-related difference in the alpha coefficients of the fitted induction curves was observed for gamma-ray-exposed lymphocytes obtained from four donors whose ages ranged from 24 to 79 years, or for tritium beta-ray-exposed lymphocytes from two donors aged 36 and 62 years. Duplicate samples from one donor, irradiated concurrently at 20 degrees C or 37 degrees C, gave significantly different alpha coefficients: 0.128+/-0.008 and 0.053+/-0.004, respectively (p<0.0001). The S-ratio (the ratio of induced complete to incomplete translocations) was found to be independent of radiation dose, donor age and exposure temperature. CONCLUSIONS: For biodosimetry in chronic irradiation situations, the use of alpha coefficients derived from the dose-response curves of cells chronically irradiated in vitro at body temperature is recommended. With respect to induction rates, donor age does not appear to be a confounding factor. The S-ratio is independent of radiation doses, exposure temperatures, or donor ages.

Biological dosimetry of beta-ray exposure from tritium using chromosome translocations in human lymphocytes analyzed by fluorescence in situ hybridization.

Radiation exposures from tritium make up a substantial fraction of the occupational and accidental radiation exposures associated with the nuclear power industry. Tritiated water, the most abundant form of tritium, is of particular interest because it is readily taken up by human cells and its irradiation of the cells is spread over a period of days. To approximate the prolonged exposure and the conditions that the cells of an individual would experience in vivo, we irradiated human lymphocytes with tritiated water for 48 h in a 1:1 blood:medium mix. For estimation of the tritium beta-ray dose, a cellular water content of 0.78, based on measurements of human lymphoblastoid cells in culture medium, was used. A modified dose calculation formula was developed for the radiation exposure conditions. A total of 48,014 metaphases (14,482 in irradiated samples and 33,532 in control, unirradiated samples) in human lymphocytes cultured for 72 h after exposure were analyzed for chromosome translocations using fluorescence in situ hybridization. The linear slope (alpha coefficient) of the dose-response curve was measured to be (3.93+/-0.42) x 10(-2) and (5.26+/-0.48) x 10(-2) translocations per cell per gray for complete translocations (tc) and complete translocations plus incomplete translocations [ti(Ab)], respectively, when the data were fitted to a linear model using a weighted least-squares method. The alpha coefficient for tc is significantly lower than that for conventionally measured dicentrics after tritium beta irradiation, but the alpha coefficient for tc + ti(Ab) does not differ significantly from that for dicentrics. This is in agreement with theoretical considerations. The importance of scoring criteria is stressed. The frequency of tc + ti(Ab) is proposed to be a reliable biodosimeter for tritium exposures, and its practical use in a dose reconstruction is presented.

Accidental intake of tritiated water: a cytogenetic follow-up case on translocation stability and dose reconstruction.

PURPOSE: To examine by fluorescence in situ hybridization (FISH) chromosomal translocations in a person who, 11 years previously, had accidentally incorporated tritiated water. To compare the resultant estimate of radiation dose with contemporary dosimetry made by urine analysis and dicentric chromosome scoring. MATERIALS AND METHODS: Blood lymphocytes were shared by two laboratories each performing the FISH analysis using different chromosome probe combinations. Doses were calculated by reference to an in vitro calibration curve produced in one of the laboratories. RESULTS: Good agreement in translocation yields was found by the two laboratories. Comparing the yields with the dicentric frequency obtained shortly after the accident and with a translocation frequency measured 6 years post exposure showed good agreement between all measurements. This indicates essentially perfect stability for translocations over an 11 year period in this individual. CONCLUSIONS: Dose reconstruction based on FISH-measured translocations showed good agreement with the dose estimated from initial dicentric measurements and from measurements of tritium in urine. Because of the extensive initial dosimetry performed on this individual, who received a uniform whole-body irradiation, the case serves as an excellent test for the use of FISH-measured translocations for retrospective biodosimetry.

Cytogenetic signature for ionizing radiation.

A stable, easily measurable biological 'signature' for past exposure to densely ionizing radiation would be of significant value to estimate environmental radiation risk. Defined here, the S-ratio is the ratio of complete to incomplete chromosome translocations. It constitutes a clastogenic signature that appears to be stable over time, independent of dose, and varies inversely with the relative biological effectiveness (RBE) of the radiation. In vitro measurements resulted in a ratio of approximately 2 for densely ionizing radiation (56Fe and 12C ions) in contrast to a value of approximately 10 for sparsely ionizing radiation (X- and gamma-rays). The measured ratio was approximately 2 for bleomycin and benzene. Such a distinctive clastogenic signature should facilitate better estimates of neutron dose for A-bomb survivors, as well as a causal connection between early exposure to densely ionizing radiation and late development of cancer.

Dose reconstruction for individuals exposed to ionizing radiation using chromosome painting.

To be most useful, a biomarker for dose reconstruction should employ an end point that is highly quantitative, stable with time and easily measured. Reciprocal translocations have been shown to be a promising biomarker that is linked to both prior exposure and risk, and they can be measured easily and quantitatively using fluorescence in situ hybridization. In contrast to other biomarkers that are available, the frequency of reciprocal translocations in individuals exposed to whole-body radiation is stable with time after exposure, has rather small interindividual variability and can be measured accurately at low levels of exposure. Results from recent studies demonstrate that measurements of reciprocal translocation frequencies, facilitated by chromosome painting, can be used to reconstruct radiation dose for individuals exposed in the distant past.

Computer simulation of data on chromosome aberrations produced by X rays or alpha particles and detected by fluorescence in situ hybridization.

With fluorescence in situ hybridization (FISH), many different categories of chromosome aberrations can be recognized-dicentrics, translocations, rings and various complex aberrations such as insertions or three-way interchanges. Relative frequencies for the various aberration categories indicate mechanisms of radiation-induced damage and reflect radiation quality. Data obtained with FISH support a proximity version of the classic random breakage-and-reunion model for the formation of aberrations. A Monte Carlo computer implementation of the model, called the CAS (chromosome aberration simulator), is generalized here to high linear energy transfer (LET) and compared to published data for human cells irradiated with X rays or 238Pu alpha particles. For each kind of radiation, the CAS has two adjustable parameters: the number of interaction sites per cell nucleus and the number of reactive double-strand breaks (DSBs) per gray. Aberration frequencies for various painted chromosomes, of varying lengths, and for 11 different categories of simple or complex aberrations were simulated and compared to the data. The optimal number of interaction sites was found to be approximately 13 for X irradiation and approximately 25 for alpha-particle irradiation. The relative biological effectiveness (RBE) of alpha particles for the induction of reactive DSBs (which are a minority of all DSBs) was found to be approximately 4. The two-parameter CAS model adequately matches data for many different categories of aberrations. It can use data obtained with FISH for any one painting pattern to predict results for any other kind of painting pattern or whole-genome staining, and to estimate a suggested overall numerical damage indicator for chromosome aberration studies, the total misrejoining number.

A rapid method for measuring pericentric inversions using fluorescence in situ hybridization (FISH).

We used three common fluorescent probes to measure pericentric inversion frequencies in 2.9 Gy 60Co gamma-irradiated human lymphocytes. For a given chromosome, the first probe is specific to one telomeric region, the second probe is specific to one subcentromeric region and the third probe is specific to the centromere. A pericentric inversion is made observable by the change in position (switching) of the fluorescent signals relative to the chromosome centromere. Our data showed equality between pericentric inversions and centric rings. The calculated whole-genome F-ratio of apparently simple translocations to pericentric inversions was 5.6.

Emerging technological bases for retrospective dosimetry.

In this article we discuss examples of challenging problems in retrospective dosimetry and describe some promising solutions. The ability to make measurements by accelerator mass spectrometry and luminescence techniques promises to provide improved dosimetry for regions of Belarus, Ukraine and Russian Federation contaminated by radionuclides from the Chernobyl accident. In addition, it may soon be possible to resolve the large neutron discrepancy in the dosimetry system for Hiroshima through novel measurement techniques that can be used to reconstruct the fast-neutron fluence emitted by the bomb some 51 years ago. Important advances in molecular cytogenetics and electron paramagnetic resonance measurements have produced biodosimeters that show potential in retrospective dosimetry. The most promising of these are the frequency of reciprocal translocations measured in chromosomes of blood lymphocytes using fluorescence in situ hybridization and the electron paramagnetic resonance signal in tooth enamel.