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Purpose: Sodium butyrate is known to inhibit histone deacetylase enzymes and to enhance the frequencies of X-ray-induced dicentrics and rings in human lymphocytes. In this study an investigation was made of the mechanisms underlying this enhancement by assessing the effect of sodium butyrate on the extent of X-ray-induced DNA damage and its repair in human peripheral blood lymphocytes. Methods and materials: Unstimulated G[sub 0] lymphocytes were pre-treated for 24h with sodium butyrate at a final concentration of 5 mM, irradiated with different doses of X-rays and then analysed for different endpoints either immediately or after different repair periods. The frequencies of DNA strand breaks were determined biochemically using nucleoid sedimentation, alkaline elution and immunochemical analysis as well as cytogenetically using the premature chromosome condensation (PCC) technique. Results: The results show that sodium butyrate pre-treatment does not lead to a significant increase of DNA double- or single-strand breaks nor to an increase of alkali labile base damage in G[sub 0] lymphocytes. Moreover, sodium butyrate treatment had no effect on the initial frequency of chromosome breaks. However, PCC analysis clearly showed that the presence of sodium butyrate post-irradiation severely inhibited DNA double-strand break (DSB) repair, which most likely accounts for the increase in X-ray-induced chromosome aberrations. Conclusions: Sodium butyrate treatment leading to changes in histone acetylation and increased accessibility of chromatin had no effect on the initial levels of X-ray-induced DNA damage. However, sodium butyrate may affect either the chromatin configuration or the enzymatic activities that play a key role in the repair of DSB. [ABSTRACT FROM AUTHOR]

Purpose: To generate dose-response curves for X-ray-induced chromosomal aberrations analysed in human blood lymphocytes using telomeric and centromeric peptide nucleic acid (PNA) probes. Materials and methods: Isolated human lymphocytes were X-irradiated with doses of 0, 1, 2, 3, 4 and 6 Gy. Aberrations were analysed in the first post-irradiation metaphases using telomeric and centromeric PNA probes. Results: Similar to the dose-response curves for the yield of dicentrics and centric rings, the dose-response curves for interstitial fragments and incomplete elements (derived from either terminal deletions or incomplete exchanges) follow a linear-quadratic function. Furthermore, it was estimated that 76% of excess acentric fragments originate from complete exchanges (interstitial deletions) and only 24% from incomplete exchanges or terminal deletions. Conclusions: Interstitial fragments form a major class of radiation-induced chromosomal aberrations. They are induced about half as frequently as dicentrics over the whole dose range investigated. The comparable trend of the dose-response curve for the different aberrations, including incomplete elements, indicates that all detected aberrations are formed by a similar underlying mechanism. It also suggests that the ratio between non- or incomplete repair (leading to open ends of broken chromosomes) and incorrect repair (leading to exchange aberrations) is independent of dose. [ABSTRACT FROM AUTHOR]

Purpose: To study by fluorescence in situ hybridization (FISH) the involvement of Chinese hamster chromosomes 3, 4, 8, and 9, and their separate chromosome arms in X-ray-induced aberrations. Materials and methods: Male embryonic primary cells of Chinese hamster were used and metaphases were collected and scored at 20h after confluent cells were exposed to 1 and 4Gy X-rays. The frequencies and types of chromosomal aberrations involving chromosomes 3, 4, 8, and 9 were studied by FISH using armspecific painting probes. Proportional distribution of the induced aberrations was tested on the basis of the relative lengths of chromosomes or chromosome arms studied. Results: A non-proportional distribution of breaks, colour junctions as well as apparently simple dicentrics and translocations among the chromosomes studied was observed in the 4Gy group. In general, chromosome 3 was less involved than expected and chromosome 8 was more involved than expected, and chromosomes 4 and 9 were involved as expected. A non-proportional involvement of arms in breaks, colour junctions and apparently simple dicentrics was also observed. The short arm of chromosome 3 was more involved in breaks and colour junctions than expected. The long arm of chromosome 4 was more involved in dicentrics than expected. Conclusions: Results of this study indicate a non-proportional involvement of Chinese hamster chromosomes 3, 4, 8 and 9 as well as their arms in different types of aberrations following irradiation. [ABSTRACT FROM AUTHOR]

Purpose : To study the frequencies and distribution of X-rayinduced chromosomal aberrations in different arms as well as diff erent regions of the long arms of Chinese hamster X chromosomes by fluorescence in situ hybridization (FISH). Material and methods : Female embryonic primary cells of Chinese hamster were exposed to 1 and 4Gy X-rays during the G1 stage and metaphases were collected 20h later with colcemid-blocking. Induced aberrations involving diff erent arms as well as different regions of the long arm of the X chromosomes were analysed by three-colour FISH using arm-specific painting probes. Distributions of aberrations in the arms were statistically analysed by chi 2-test, with the hypothesis that aberrations are proportionally distributed on the basis of their relative lengths of the arms. Results : The long arms of the X chromosomes were frequently involved in breaks than was the short arm. The result of the chi 2- test indicates a non-proportional distribution of breaks between the arms, while exchanges (dicentrics and translocations) involving the arms were proportionally distributed. Differential involvement of regions of the long arm, i.e. Xq1 and Xq2, in breaks was also observed. Xq21, a known common fragile site in the X chromosome, was often involved in terminal deletions. Conclusion : Arm-specific probes of Chinese hamster chromosomes are useful for the detailed study of X-ray-induced aberrations in the X chromosome. The heterogeneity of the Chinese hamster X chromosome in response to X-ray-induced aberrations exists not only between the short (euchromatin) and the long (heterochromatin) arms, but also between different heterochromatic regions of the long arm of the X chromosome. [ABSTRACT FROM AUTHOR]

Purpose: To investigate the induction and persistence of chromosome aberrations in mouse bone marrow cells after X-ray exposure and to detect differential involvement of individual chromosomes in translocations. Materials and methods: Male and female Swiss mice were exposed to 1 and 3Gy of X-rays. Chromosome aberrations in bone marrow cells were analysed at 1, 7, 21 and 100 days following irradiation by means of fluorescence in situ hybridization (FISH) with mouse chromosome-specific DNA libraries (#1,13; #2,8; #6,15 and X,Y). In total, 38% of mouse genome was painted and examined. Results: Pooled data indicate that the frequencies of dicentrics and fragments decreased with time and reached to the control level at day 21 after exposure. Following exposure to 1Gy of Xrays, the frequencies of translocations were not significantly lower between days 7 and 100 than observed at day 1. However, the frequencies of translocations for the 3Gy group were significantly (about 40%) lower at day 7, then remained constant up to day 100. After exposure to 3Gy of X-rays, the frequencies of non-reciprocal translocations decreased with time, whereas reciprocal translocations between days 7 and 100 were not significantly less frequent than at day 1. A comparison of observed and expected numbers of translocations involving individual chromosomes showed that at day 1 after irradiation, distribution of X-ray-induced translocations among the painted chromosomes was proportional to their DNA content. However, at day 100 after exposure, the observed translocations involving chromosome 2 were more frequent than expected, those involving chromosomes 8 and 15 were less frequent than expected, while chromosomes 1, 6, X and Y were involved as frequently as expected. Conclusion: Among induced translocations, non-reciprocal translocations are relatively unstable, especially after exposure to high-dose X-rays. While the initial distribution of X-ray-induced translocations is proportional among the painted chromosomes, the persistence of these translocations is heterogeneous. [ABSTRACT FROM AUTHOR]

Purpose: To develop a mouse model for retrospective biological dosimetry, the kinetics of induction and persistence of chromosome aberrations following X-irradiation are analysed. Materials and Methods: Female Swiss mice were whole-body exposed to 0, 0.5, 1.0, 2.0 or 3.0 Gy X-rays. Fluorescence in situ hybridization using composite DNA libraries specific for chromosomes 1, 11 and 13 was performed on metaphase chromosomes from isolated splenocytes at 0, 7, 28, 56, 112 and 224 days post irradiation to detect chromosome aberrations. Results: Dose-response curves for all types of aberrations observed \[dicentrics, fragments, metacentrics (Robertsonian fusion-like configurations) and translocations] were well fitted by a linear quadratic model. Equal induction of dicentrics and translocations was observed immediately after irradiation for all the doses used. The frequency of dicentrics and fragments decayed exponentially with time. Genomic translocation frequency declined with time post-irradiation and reached stability during later intervals, which was strongly dose-dependent. Reciprocal translocations and incomplete exchanges (terminal translocations) were induced at almost equal frequency immediately after X-irradiation (exception 3.0 Gy) and showed different patterns of decline. Conclusion: The results, on the decay of genomic translocation frequency with time after irradiation in mouse splenocytes, have implications on the use of translocations in retrospective biological dosimetry. [ABSTRACT FROM AUTHOR]

Purpose: Analysis of chromosomal aberrations by fluorescence in situ hybridization using a combination of chromosome painting and telomere detection in order to get more insight into: (a) the extent of incompleteness of exchanges and (b) the frequencies of interstitial fragments. Materials and methods: Isolated mouse splenocytes were exposed in vitro to X-rays at a dose of 2 Gy. Aberrations involving chromosomes 2 and 3 were analysed by FISH using simultaneous chromosome painting and telomere detection. Results: At 2 Gy, about 10% of apparently simple exchanges are incomplete. A striking observation was the high induction of interstitial fragments, with frequencies nearly as high as that of dicentrics. Assuming, that both ends of all interstitial fragments have rejoined with each other (to form acentric rings), it can be estimated that over 92% of reactive ends of detectable breakpoints have rejoined illegitimately. Overall, equal frequencies of translocation types t(Ab) and t(Ba) (according to the PAINT nomenclature) were observed. Also, the ratios between reciprocal forms of translocations and dicentrics were close to 1 for both the chromosomes studied. Conclusions: These studies have shown that many of the frequently observed 'one-way' exchanges using painting probes, are in fact reciprocal exchanges with one participating lesion so close to the telomere that no distal signal can be detected. Frequencies of true incomplete exchanges were found to be low. Intrachanges, here detected as interstitial fragments, were observed frequently. [ABSTRACT FROM AUTHOR]

Purpose: To estimate the frequencies of radiation- (low and high LET) induced chromosome aberrations in Chinese hamster splenocytes by two-colour fluorescence in situ hybridization using DNA painting probes specific for chromosomes 2, 3, 8, X and Y and to determine (1) the ratio of radiation-induced translocations and dicentrics; (2) the spectrum of exchange aberrations induced by X-rays and neutrons; and (3) the relative involvement of the different chromosomes in the formation of aberrations. Materials and methods: Isolated splenocytes from the Chinese hamster were irradiated in vitro with different doses of 200kV X-rays (0.75, 1.5, 3.0Gy) and 1MeV fast neutrons (0.25, 0.5, 1.0Gy). Conventional analysis of chromosome aberrations was carried out in Giemsa-stained preparations. Chromosome aberrations involving chromosomes 2, 3, 8, X and Y were analysed in first division metaphases using two-colour FISH. Results: The results indicate that when all types of translocations are taken into account both X-rays and neutrons induce more translocations than dicentrics, the ratio between the two types of exchanges being 1.4 and 1.8 respectively. The ratio of 'apparently simple' reciprocal translocations and reciprocal complete dicentrics was close to 1 for both types of radiation. The RBE of neutrons for induction of exchanges was found to be between 5 and 8. Neutron irradiation was more efficient at inducing insertions. Among the chromosomes studied, an increased involvement was observed for chromosome 8 in dicentrics and translocations than that expected on the basis of its chromosome length. The high content of interstitial telomeric sequences in chromosome 8 may be responsible for the observed sensitivity of this chromosome. Conclusions: The results obtained in this study indicate that: (1) more translocations are found than dicentrics; (2) heterogeneity exists among Chinese hamster chromosomes for involvement in radiation-induced exchanges; (3) the spectrum and distribution of exchange aberrations are different between X-rays and neutrons; and (4) the relative frequencies of insertions could be used as a 'fingerprint' for exposure to high LET radiation. [ABSTRACT FROM AUTHOR]

Abstract. Whole blood samples from two healthy donors were cultured in the presence of 5-bromo-2-deoxyuridine (BrdU) for a total of 107 h following in vitro X-irradiation with a dose of 2 Gy. Starting from 35 h after culture initiation, every subsequent 12 h a sample was taken from each culture and grown in the presence of demecolcine for another 12 h. At each sampling time, the aberrations involving chromosomes 1 and 4 were analysed using dual-colour fluorescence in situ hybridization (FISH) with chromosome-specific DNA libraries. Following differential staining of sister chromatids, the analysed cells were identified to be either in their first, second or third etc. mitosis after irradiation. Cells within the same postirradiation division contained higher frequencies of aberrations when derived from later sampling times, indicating a delay in progression of aberrant cells to mitosis. In contrast, when the aberration frequencies are calculated by sampling time (i.e. independent of the cell cycle) minimal effect of sampling time could be seen. This observation held true for all types of chromosomal aberrations. Analysis of about 2250 first-division cells for each donor (derived from all sampling times) indicates a relative overrepresentation of chromosome 4 in the formation of exchange aberrations/colour junctions. Whereas dicentric frequencies for chromosomes 1 and 4 were close to the expected values based on the DNA content of these chromosomes, frequencies of reciprocal translocations showed a clear overinvolvement of chromosome 4. This resulted in a distinct difference in the reciprocal translocation to dicentric ratio, being 1.12 for chromosome 1 and 2.09 for chromosome 4. These results indicate a non-DNA-proportional distribution of radiation-induced chromosome rearrangements in cultured human lymphocytes. [ABSTRACT FROM AUTHOR]

Focuses on the discrimination between radiation-induced complete and incomplete chromosome exchanges using chromosome painting. Detection of centromeres and telomeres in fluorescence in situ hybridization (FISH) assay; Impact of the combined FISH Assay on lymphocytes; Observation of low fraction of true incomplete exchanges in lymphocytes.

Purpose: In mammalian cells, the relevance of homologous recombination in radiation-induced double-strand break (DSB) repair is not yet well understood. In the present work, the role of recombination between homologous chromosomes and homology-directed repair of DSB were studied, using X-ray-induced chromosomal aberrations as an end-point., Materials and Methods: Human-hamster hybrid cells containing one or two copies of human chromosome 8 were used. If recombination between homologous chromosomes plays a dominant role in DSB repair, it is expected that X-irradiation of cells with two copies of chromosome 8 would result in a lower frequency of aberrations involving this chromosome compared with cells with only one copy of chromosome 8. The aberrations involving human chromosome 8 were detected by fluorescence in situ hybridization (FISH). Furthermore, a comparison between the hamster cell line XR-C1 (defective in non-homologous repair), CHO-9 (the wild-type cells) and the cell line XR-C1#8 (in which the defect of XR-C1 is complemented by human chromosome 8) was made to determine, indirectly, the involvement of homology-directed recombination in DSB repair., Results: The observed frequencies of aberrations per human chromosome 8 were not significantly different between cells containing one or two copies of this chromosome. The frequency of chromatid-type aberrations was doubled in XR-C1 cells compared with CHO-9 and XR-C1#8 cells., Conclusions: In hamster cells, recombination between homologous chromosomes appears not to have a major role in the formation of radiation-induced chromosomal aberrations, while nonhomologous repair seems to be important in both the G and G2 phases of the cell cycle.

The frequencies of chromosome aberrations induced by different doses of X-rays were determined in both splenocytes and primary lung fibroblasts of Chinese hamster by bi-colour FISH using a combination of four chromosome-specific DNA libraries. The results indicate that the X-rays induced more translocations than dicentrics in Chinese hamster cells, in which the karyotype is comprised of both metacentric and acrocentric chromosomes. These results are similar to those reported in human lymphocytes, in which the karyotype contains many metacentric chromosomes. On the contrary, in mouse, which is characterized by acrocentric chromosomes only, the frequencies of translocations and dicentrics are induced in nearly equal proportions by X-rays. The ratio of translocations to dicentrics obtained in Chinese hamster cells was approximately 1.4-1.5, which supports the importance of the karyotypic features of a species in the relative induction of translocations to dicentrics. An analysis was also made on the yield of translocations and dicentrics involving individual chromosomes and the results indicate a non-random involvement of these chromosomes in the formation of aberrations.

A characteristic karyotypic feature of mouse chromosomes is the presence of large blocks of heterochromatin in the vicinity of the centromeres. Breakage inside this centromeric heterochromatin might result in the formation of abnormal chromosomes, very similar to the metacentric chromosomes derived from Robertsonian fusion. X-rays are very efficient in inducing these Robertsonian fusion-like configurations (RLC) in cultured mouse splenocytes. Observed frequencies of these RLC increase in a linear-quadratic manner with dose. Two types of RLC were found. The first type (approximately 70% of induced RLC) has heterochromatin only in the middle of the chromosome and appears as a metacentric chromosome, whereas the other type (approximately 30%) has a heterochromatic block inside the chromosome arm (and has the appearance of a dicentric chromosome). Induced RLC are difficult to classify as either a stable or unstable aberration, based only on traditional cytogenetic techniques such as C-banding. Here we describe a cytogenetic approach to achieving better insight into the molecular organization of RLC. We utilized two-colour fluorescence in situ hybridization (FISH) using a combination of mouse minor satellite DNA probe and telomeric probe. Over 90% of RLC did not have detectable minor satellite arrays inside the interstitial heterochromatin. Consequently, most of the RLC of the first type should be classified as acentric fragments and those of the second type as translocations. The chance of inducing true Robertsonian fusions in mouse splenocytes by X-rays is < 2.5% based on the total RLC observed.

Chromosome translocations (stable aberrations) can persist longer during cellular proliferation than dicentrics (unstable aberrations). It is important to know the kinetics of the elimination of dicentrics and to what extent translocations persist in an in vivo cell population after irradiation. The female Swiss mouse were used to study the induction and persistence of dicentrics and translocations in splenocytes up to 112 days after exposure to 2 Gy whole-body X-irradiation. Metaphase spreads at different time intervals were analyzed by fluorescence in situ hybridization (FISH) using chromosome-specific DNA libraries for chromosomes 1, 11 and 13. The frequencies of dicentrics and translocations appear to be equal immediately after irradiation. Frequencies of dicentrics decreased exponentially with time according to the relationship D = ae(-kt). The rate of elimination was faster in the early period (days 0-14) than in the later period (> or = 14 days). The frequency of translocations was constant in the period 0-7 days and then decreased linearly or exponentially. For the whole period, the trend is highly significant. As mouse chromosome painting probes are becoming available and by using FISH, an in vivo mouse model for the analysis of translocations has become feasible. As translocations are involved in carcinogenesis and genetic disorders, risk estimation for induction of translocations by ionizing radiation can be made with greater confidence and extrapolated to the human situation.

A total of 255 chromosomal aberrations induced by X-rays in G1 phase of the cell cycle were scored in 600 G-banded metaphases prepared from Chinese hamster female cells. On the basis of a detailed analysis of these aberrations a total of 441 chromosomal breakpoints were mapped to the individual Chinese hamster chromosomes and their bands. More breakpoints were mapped to G-light (80.5%) than to G-dark (19.5%) bands. Chromosomal distribution of breakpoints revealed that chromosomes 5 and 8 had significantly more exchange breakpoints than expected on the basis of chromosomal length, whereas the X chromosome had significantly more terminal deletion breakpoints than expected. At the same time chromosomes 5 and 8 had a deficiency of terminal deletions, whereas the X chromosome had significant deficiency of exchange breakpoints. These results indicate that radiation-induced exchange and terminal deletion breakpoints, as observed in the first postirradiation metaphase, have different patterns of distribution in Chinese hamster chromosomes. Clustering of terminal deletions in the long arms of X chromosomes, which are entirely occupied by heterochromatin, suggests that chromosomal repair mechanisms responsible for rejoining of chromosomal breaks are less effective in heterochromatic than in other genomic regions.