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Cancer risk after ionizing radiation (IR) is assumed to be linear with the dose; however, for low doses, definite evidence is lacking. Here, using temporal multi-omic systems analyses after a low (LD; 0.1 Gy) or a high (HD; 1 Gy) dose of X-rays, we show that, although the DNA damage response (DDR) displayed dose proportionality, many other molecular and cellular responses did not. Phosphoproteomics uncovered a novel mode of phospho-signaling via S12-PPP1R7, and large-scale dephosphorylation events that regulate mitotic exit control in undamaged cells and the G2/M checkpoint upon IR in a dose-dependent manner. The phosphoproteomics of irradiated DNA double-strand breaks (DSBs) repair-deficient cells unveiled extended phospho-signaling duration in either a dose-dependent (DDR signaling) or independent (mTOR-ERK-MAPK signaling) manner without affecting signal magnitude. Nascent transcriptomics revealed the transcriptional activation of genes involved in NRF2-regulated antioxidant defense, redox-sensitive ERK-MAPK signaling, glycolysis and mitochondrial function after LD, suggesting a prominent role for reactive oxygen species (ROS) in molecular and cellular responses to LD exposure, whereas DDR genes were prominently activated after HD. However, how and to what extent the observed dose-dependent differences in molecular and cellular responses may impact cancer development remain unclear, as the induction of chromosomal damage was found to be dose-proportional (10-200 mGy).

Background: Preclinical evidence shows that short-term fasting (STF) protects healthy cells against side effects of chemotherapy and makes cancer cells more vulnerable to it. This pilot study examines the feasibility of STF and its effects on tolerance of chemotherapy in a homogeneous patient group with early breast cancer (BC)., Methods: Eligible patients had HER2-negative, stage II/III BC. Women receiving (neo)-adjuvant TAC (docetaxel/doxorubicin/cyclophosphamide) were randomized to fast 24 h before and after commencing chemotherapy, or to eat according to the guidelines for healthy nutrition. Toxicity in the two groups was compared. Chemotherapy-induced DNA damage in peripheral blood mononuclear cells (PBMCs) was quantified by the level of γ-H2AX analyzed by flow cytometry., Results: Thirteen patients were included of whom seven were randomized to the STF arm. STF was well tolerated. Mean erythrocyte- and thrombocyte counts 7 days post-chemotherapy were significantly higher (P = 0.007, 95 % CI 0.106-0.638 and P = 0.00007, 95 % CI 38.7-104, respectively) in the STF group compared to the non-STF group. Non-hematological toxicity did not differ between the groups. Levels of γ-H2AX were significantly increased 30 min post-chemotherapy in CD45 + CD3- cells in non-STF, but not in STF patients., Conclusions: STF during chemotherapy was well tolerated and reduced hematological toxicity of TAC in HER2-negative BC patients. Moreover, STF may reduce a transient increase in, and/or induce a faster recovery of DNA damage in PBMCs after chemotherapy. Larger studies, investigating a longer fasting period, are required to generate more insight into the possible benefits of STF during chemotherapy., Trial Registration: ClinicalTrials.gov: NCT01304251 , March 2011.

DNA lesions, induced by genotoxic compounds, block the processive replication fork but can be bypassed by specialized translesion synthesis (TLS) DNA polymerases (Pols). TLS safeguards the completion of replication, albeit at the expense of nucleotide substitution mutations. We studied the in vivo role of individual TLS Pols in cellular responses to benzo[a]pyrene diolepoxide (BPDE), a polycyclic aromatic hydrocarbon, and 4-hydroxynonenal (4-HNE), a product of lipid peroxidation. To this aim, we used mouse embryonic fibroblasts with targeted disruptions in the TLS-associated Pols η, ι, κ, and Rev1 as well as in Rev3, the catalytic subunit of TLS Polζ. After exposure, cellular survival, replication fork progression, DNA damage responses (DDR), and the induction of micronuclei were investigated. The results demonstrate that Rev1, Rev3, and, to a lesser extent, Polη are involved in TLS and the prevention of DDR and of DNA breaks, in response to both agents. Conversely, Polκ and the N-terminal BRCT domain of Rev1 are specifically involved in TLS of BPDE-induced DNA damage. We furthermore describe a novel role of Polι in TLS of 4-HNE-induced DNA damage in vivo. We hypothesize that different sets of TLS polymerases act on structurally different genotoxic DNA lesions in vivo, thereby suppressing genomic instability associated with cancer. Our experimental approach may provide a significant contribution in delineating the molecular bases of the genotoxicity in vivo of different classes of DNA-damaging agents.

The recent steep increase in population dose from radiation-based medical diagnostics, such as computed tomography (CT) scans, requires insight into human health risks, especially in terms of cancer development. Since the induction of genetic damage is considered a prominent cause underlying the carcinogenic potential of ionizing radiation, we quantified the induction of micronuclei and loss of heterozygosity events in human cells after exposure to clinically relevant low doses of X rays. A linear dose-response relationship for induction of micronuclei was observed in human fibroblasts with significantly increased frequencies at doses as low as 20 mGy. Strikingly, cells exposed during S-phase displayed the highest induction, whereas non S-phase cells showed no significant induction below 100 mGy. Similarly, the induction of loss of heterozygosity in human lymphoblastoid cells quantified at HLA loci, was linear with dose and reached significance at 50 mGy. Together the findings favor a linear-no-threshold model for genetic damage induced by acute exposure to ionizing radiation. We speculate that the higher radiosensitivity of S-phase cells might relate to the excessive cancer risk observed in highly proliferative tissues in radiation exposed organisms.

In interphase, chromosomes occupy defined nuclear volumes known as chromosome territories. To probe the biological consequences of the described nonrandom spatial positioning of chromosome territories in human lymphocytes, we performed an extensive FISH-based analysis of ionizing radiation-induced interchanges involving chromosomes 1, 4, 18 and 19. Since the probability of exchange formation depends strongly on the spatial distance between the damage sites in the genome, a preferential formation of exchanges between proximally positioned chromosomes is expected. Here we show that the spectrum of interchanges deviates significantly from one expected based on random chromosome positioning. Moreover, the observed exchange interactions between specific chromosome pairs as well as the interactions between homologous chromosomes are consistent with the proposed gene density-related radial distribution of chromosome territories. The differences between expected and observed exchange frequencies are more pronounced after exposure to densely ionizing neutrons than after exposure to sparsely ionizing X rays. These experiments demonstrate that the spatial positioning of interphase chromosomes affects the spectrum of chromosome rearrangements.

We previously reported that exposure of human cells to DNA-damaging agents (X-rays and mitomycin C (MMC)) induces pairing of the homologous paracentromeric heterochromatin of chromosome 9 (9q12-13). Here, we show that UV irradiation and also heat shock treatment of human cells lead to similar effects. Since the various agents induce very different types and frequencies of damage to cellular constituents, the data suggest a general stress response as the underlying mechanism. Moreover, local UV irradiation experiments revealed that pairing of heterochromatin is an event that can be triggered without induction of DNA damage in the heterochromatic sequences. The repair deficient xeroderma pigmentosum cells (group F) previously shown to fail pairing after MMC displayed elevated pairing after heat shock treatment but not after UV exposure. Taken together, the present results indicate that pairing of heterochromatin following exposure to DNA-damaging agents is initiated by a general stress response and that the sensing of stress or the maintenance of the paired status of the heterochromatin might be dependent on DNA repair.

Chromatid interchanges induced by the DNA cross-linking agent mitomycin C (MMC) are over-represented in human chromosomes containing large heterochromatic regions. We found that nearly all exchange breakpoints of chromosome 9 are located within the paracentromeric heterochromatin and over 70% of exchanges involving chromosome 9 are between its homologues. We provide evidence that the required pairing of chromosome 9 heterochromatic regions occurs in G(0)/G(1) and S-phase cells as a result of an active cellular process initiated upon MMC treatment. By contrast, no pairing was observed for a euchromatic paracentromeric region of the equal-sized chromosome 8. The MMC-induced pairing of chromosome 9 heterochromatin is observed in a subset of cells; its percentage closely mimics the frequency of homologous interchanges found at metaphase. Moreover, the absence of pairing in cells derived from XPF patients correlates with an altered spectrum of MMC-induced exchanges. Together, the data suggest that the heterochromatin-specific pairing following MMC treatment reflects the initiation of DNA cross-link repair and the formation of exchanges.

The human-Chinese hamster hybrid cell line XR-C1#8, containing human chromosome 8, was used as a model system to investigate the relative importance of cellular enzymatic environment and chromosomal structure for modulating the efficiency of repair of UV-induced DNA damage. The hybrid cells were irradiated with UVC light and the extent of cytogenetic damage, detected as frequencies of sister chromatid exchanges (SCEs), was compared between the human and the hamster chromosomes. The combination of immunofluorescent staining for SCEs and chromosome painting with fluorescence in situ hybridization allowed the simultaneous analysis of SCEs in the human and hamster chromosomes. The aim of the present study was to determine if the differences in biological response to comparable UV treatments observed between human and hamster cells were maintained in the hybrid cells in which human and hamster chromosomes are exposed in the same cellular environment. The analysis of replication time of human chromosome 8 indicated the active status of this chromosome in XR-C1#8 hybrid cells. The frequencies of SCEs for human chromosome 8 and a hamster chromosome of comparable size were 0.35 +/- 0.52, 0.80 +/- 0.73, 1.24 +/- 2.24 and 0.36 +/- 0.12, 0.71 +/- 0.2, 0.97 +/- 0.27, respectively, after irradiation with 0, 5, and 10 J/m2. The persistence of UV-induced SCEs after three cell cycles was also analyzed, both for the human and hamster chromosomes. The observed frequencies of SCEs were 0.40 +/- 0.57, 0.62 +/- 1.05, 0.58 +/- 0.83 and 0.26 +/- 0.08, 0.67 +/- 0.18, 0.69 +/- 0.24, in human and hamster chromosomes respectively, after treatment with 0, 10, and 20 J/m2 of UVC light. No significant differences could be observed between the human and hamster chromosomes. These results suggest that the enzymatic environment of human and hamster cells has the main role, in comparison to the structural organization of human and hamster chromosomes, for determining the different level of repair of UV-induced DNA damage observed in these two species., (Copyright 2003 S. Karger AG, Basel)

Using fluorescence in situ hybridization with human band-specific DNA probes we examined the effect of ionizing radiation on the intra-nuclear localization of the heterochromatic region 9q12-->q13 and the euchromatic region 8p11.2 of similar sized chromosomes 9 and 8 respectively in confluent (G1) primary human fibroblasts. Microscopic analysis of the interphase nuclei revealed colocalization of the homologous heterochromatic regions from chromosome 9 in a proportion of cells directly after exposure to 4 Gy X-rays. The percentage of cells with paired chromosomes 9 gradually decreased to control levels during a period of one hour. No significant changes in localization were observed for chromosome 8. Using 2-D image analysis, radial and inter-homologue distances were measured for both chromosome bands. In unexposed cells, a random distribution of the chromosomes over the interphase nucleus was found. Directly after irradiation, the average inter-homologue distance decreased for chromosome 9 without alterations in radial distribution. The percentage of cells with inter-homologue distance <3 micro m increased from 11% in control cells to 25% in irradiated cells. In contrast, irradiation did not result in significant changes in the inter-homologue distance for chromosome 8. Colocalization of the heterochromatic regions of homologous chromosomes 9 was not observed in cells irradiated on ice. This observation, together with the time dependency of the colocalization, suggests an underlying active cellular process. The biological relevance of the observed homologous pairing remains unclear. It might be related to a homology dependent repair process of ionizing radiation induced DNA damage that is specific for heterochromatin. However, also other more general cellular responses to radiation-induced stress or change in chromatin organization might be responsible for the observed pairing of heterochromatic regions., (Copyright 2003 S. Karger AG, Basel)

Most of the mutagenic and carcinogenic agents induce chromosome aberrations in vivo and in vitro. Conventional solid staining (such as Giemsa) has been employed to evaluate the frequencies and types of spontaneous and induced chromosomal aberrations. Recently, molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH) using chromosome specific or chromosome region-specific DNA libraries have become available, which have increased the resolution of the detection of aberrations. This has lead to a better understanding on the mechanisms of formation of chromosome aberrations, especially following treatment with ionizing radiation. The present paper reviews briefly the results obtained using FISH technique both from basic and applied studies.

The chromosome-type exchange aberrations induced by ionizing radiation during the G(0)/G(1) phase of the cell cycle are believed to be the result of illegitimate rejoining of chromosome breaks. From numerous studies using chromosome painting, it has emerged that even after a moderate dose of radiation, a substantial fraction of these exchanges is complex. Most of them are derived from the free interaction between the ends of three or more breaks. Other studies have demonstrated that chromosomes occupy distinct territories in the interphase nucleus. Since breaks that are in close proximity have an enhanced interaction probability, it seems likely that after ionizing radiation many of the interacting breaks will be present within one chromosome or chromosome arm. Unfortunately, the majority of these intrachanges remain undetected, even when sophisticated molecular cytogenetic detection methods (i.e. mFISH) are applied to paint all chromosome pairs in distinct colors. In the present paper, we evaluate the limitations of full-color painting for the detection of complex exchanges and the correct interpretations of break interactions.

The Rev3 gene of Saccharomyces cerevisiae encodes the catalytic subunit of DNA polymerase zeta that is implicated in mutagenic translesion synthesis of damaged DNA. To investigate the function of its mouse homologue, we have generated mouse embryonic stem cells and mice carrying a targeted disruption of Rev3. Although some strain-dependent variation was observed, Rev3(-/-) embryos died around midgestation, displaying retarded growth in the absence of consistent developmental abnormalities. Rev3(-/-) cell lines could not be established, indicating a cell-autonomous requirement of Rev3 for long-term viability. Histochemical analysis of Rev3(-/-) embryos did not reveal aberrant replication or cellular proliferation but demonstrated massive apoptosis in all embryonic lineages. Although increased levels of p53 are detected in Rev3(-/-) embryos, the embryonic phenotype was not rescued by the absence of p53. A significant increase in double-stranded DNA breaks as well as chromatid and chromosome aberrations was observed in cells from Rev3(-/-) embryos. The inner cell mass of cultured Rev3(-/-) blastocysts dies of a delayed apoptotic response after exposure to a low dose of N-acetoxy-2-acetylaminofluorene. These combined data are compatible with a model in which, in the absence of polymerase zeta, double-stranded DNA breaks accumulate at sites of unreplicated DNA damage, eliciting a p53-independent apoptotic response. Together, these data are consistent with involvement of polymerase zeta in translesion synthesis of endogenously and exogenously induced DNA lesions.

Purpose: To study the impact of radiation quality on the spectrum of chromosome exchange aberrations in human lymphocytes using chromosome arm-specific and telomeric probes. The analysis is focused on: (1) incomplete exchanges, (2) interstitial fragments, (3) interarm intrachanges and (4) the complexity of the aberration patterns. The present data after neutron exposure are compared with previously obtained data after X-irradiation., Materials and Methods: Isolated human lymphocytes from three donors were irradiated with 1 MeV fast neutrons (0.25, 0.5, 1.0, 1.5, 2.0 Gy). Analysis was performed on first post-irradiation metaphases with arm-specific probes for chromosome 1 in combination with a pan-centromeric probe, or with telomeric and centromeric PNA probes., Results: In comparison with X-rays, exposure to neutrons leads to: (1) similar frequencies of incomplete exchanges or terminal deletions, (2) a significantly higher induction of both inter- and intraarm intrachanges, (3) a higher proportion of complex aberrations, and (4) aberrations with a higher degree of complexity, i.e. derived from more chromosome breaks which interact more frequently in a non-reciprocal fashion. Essentially no dose dependence was found for the yield ratios between the various types of chromosomal aberrations., Conclusions: Despite the reduced rejoining deficiency of DNA double-strand breaks induced by high-LET radiation, exposure to neutrons does not lead to enhanced levels of unrejoined chromosome breaks that can be observed as incomplete exchanges in cells that have reached mitosis. Proximity effects are more pronounced after densely ionizing radiation than after sparsely ionizing radiation. Clustered damage produced by neutron tracks results in a high proportion of complex aberrations and in non-reciprocal interactions of chromosome breaks. Most of the exchanges occur within one neutron track and little interaction seems to take place between the breaks formed in different tracks.

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.

Euchromatic and heterochromatic regions are easily distinguished in Chinese hamster sex chromosomes, hence offering the possibility of studying the role of chromatin structure in the induction, processing and persistence of radiation-induced chromosome damage. X-ray (4 Gy)-induced breaks in the euchromatic Xp and in the heterochromatic Xq were analysed immediately and 4h after irradiation by premature chromosome condensation (PCC) in combination with either FISH using chromosome arm-specific probes or Giemsa staining. The study, performed with female Chinese hamster splenocytes, was extended to a 34 h recovery followed by arm-specific FISH in metaphase. A significant over-involvement of the heterochromatic Xq in radiation-induced breakage was observed at all sampling times (p<0.001). However, the heterochromatic state had little effect on the processing of the induced lesions. In a second experiment, the persistence of radiation-induced chromosome aberrations (CAs) involving Xp, Xq and Y chromosome was studied with cultured Chinese hamster male splenocytes sampled 30, 56 and 96 h after irradiation (4 Gy). A higher involvement of the heterochromatic regions (Xq and Y) in radiation-induced CAs was again observed in the first sampling time (p<0.001), suggesting that Chinese hamster heterochromatin could be more radiosensitive than euchromatin. Cells with CAs involving heterochromatin were apparently less persistent than those with lesions involving euchromatin. This observation could be attributable to either the distribution of CA per cell or to the fraction of potentially stable exchanges.

Purpose: To discriminate precisely between radiation-induced complete and incomplete chromosome exchanges using chromosome painting together with the detection of the centromeres and telomeres in one FISH assay., Materials and Methods: Human lymphocytes were exposed in vitro to X-rays at a dose of 4 Gy. Chromosome aberrations were analysed using the FISH technique in combination with a whole chromosome-specific DNA probe for chromosome 8, human pan-centromeric DNA and telomeric PNA probes., Results: The combined FISH assay has improved the resolution of detecting chromosomal exchanges in human lymphocytes. Results indicate that the frequency of observed incomplete exchange patterns was 21% when telomeric signals were ignored during the analysis. When the telomeric signals were included in the analysis a large proportion of apparently incomplete exchange patterns appeared complete and should be re-classified. The percentage of true incomplete exchanges was found to be less than 5%., Conclusion: The combination of chromosome painting and the detection of centromeres and telomeres enable unequivocal discrimination between incomplete and complete exchanges. The fraction of true incomplete exchanges observed in X-irradiated human lymphocytes was found to be low in comparison with previous reports in the literature.

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.

Frequencies of chromosomal aberrations were estimated in X-irradiated (0; 0.5; 1; 2; 3 and 4 Gy) human lymphocytes using arm-specific probes for chromosome 1 in combination with a pancentromeric probe in a triple colour FISH procedure. This allowed the construction of dose-response curves for both interchanges as well as interarm intrachanges. We found that 11.7-18.4% of aberrant chromosomes contained interarm intrachanges. Assuming a free interaction of randomly induced exchange breakpoints throughout the whole genome, interarm intrachanges occur about 8.7 times more frequently than expected. The close proximity of the two arms of a chromosome in the interphase nucleus appears to promote the formation of interarm intrachanges. Convincing evidence was obtained, by comparing the frequency of colour junctions that occur between 1p-1q and 1p-3q (the arms 1q and 3q are of similar size). With this approach, we observed 8.8 times more colour junctions between 1p-1q than between 1p-3q, illustrating the importance of proximity in the formation of chromosomal exchange aberrations. At doses higher than 2 Gy, a saturation of simple reciprocal aberrations, both for interchanges as well as for interarm intrachanges was observed. Furthermore, we found that symmetrical and asymmetrical simple interchanges as well as interarm intrachanges occur with equal frequencies in human chromosome 1., (Copyright 1998 Elsevier Science B. V.)

Purpose: The detection of incomplete exchanges and interstitial fragments by fluorescence in situ hybridization using a telomeric peptide nucleic acid (PNA) probe., Materials and Methods: Isolated human lymphocytes were exposed in vitro to X-rays at a dose of 3 Gy. Aberrations were analysed in the first mitosis after irradiation using a telomeric PNA probe., Results: After an acute dose of 3 Gy, only about 16% of the cells contained a pair of incomplete chromosome elements with telomeric signals at only one terminal end. Acentric interstitial fragments, lacking telomeric signals, were observed with a frequency of 0.56 per cell, which was very similar to the dicentric frequency (0.61 per cell)., Conclusions: The low frequency of incompleteness suggests that most of the non-reciprocal interchanges observed using chromosome painting must originate from terminal exchanges rather than from incomplete exchanges. Furthermore, it was estimated that about 78% of excess acentric fragments originate from interstitial fragments and only 22% from terminal fragments.

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.

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.

Transgenic mice with foreign DNA inserted into three pairs of chromosomes were exposed to 2 Gy X-rays in order to study the induction and persistence of chromosome malsegregation and aneuploidy up to 28 days after exposure. By tracing the marker chromosomes in cytokinesis-blocked binucleated splenocytes using fluorescence in situ hybridization (FISH), reciprocal products of chromosome malsegregation in the daughter nuclei were analysed. FISH with murine minor satellite DNA was employed to detect chromosome loss (MN with a centromere) in binucleated splenocytes. In addition to its clastogenic effects, X-irradiation also showed aneugenic activity, which was observed as centromere positive micronuclei (C + MN) and malsegregated marker chromosomes detected by FISH. The initial frequency of micronuclei (MN) analysed by Acridine Orange staining immediately after X-ray exposure was found to be 42.3 per 100 binucleated cells. The MN frequency declined in an exponential manner and at day 14, reached about half the value observed immediately after irradiation and 14% MN were detected at day 28. Of these MN, 25% were centromere positive at day 0 as detected by minor satellite signal after FISH. The percentage C + MN increased further at day 3 and declined at day 14 to the level observed at day 0. There were 7.6% malsegregated cells immediately after X-irradiation as analysed by two colour FISH. This value increased further during later intervals and remained stable until day 28. A combination of the Acridine Orange staining and FISH with minor satellite DNA and marker DNA to detect aneuploidy and chromosome malsegregation, was utilized in the present study to demonstrate the induction and persistence of aneugenic and clastogenic damage in transgenic mice irradiated in vivo.

Fluorescence in situ hybridization (FISH) technique using chromosome specific probes has revolutionized the field of radiation cytogenetics in the last few years. Some of the new insights on the origins of radiation induced chromosome aberrations in human, mouse and Chinese hamster, using FISH are reviewed in this paper.

Frequencies of intra-chromosomal exchanges (pericentric inversions and centric rings) and inter-chromosomal exchanges (dicentrics and translocations) in X-irradiated (2.5 Gy) human lymphocytes have been estimated. To detect these events we employed FISH (fluorescence in situ hybridization) technique and arm specific painting probes for chromosomes #1 and #3. The ratio between centric rings and pericentric inversions was found to be about 1. For intra-changes to inter-changes, the ratio (F) was between 6 and 9. Based on the total number of colour junctions involving chromosomes #1 and #3 it was found that exchanges between the arms of the same chromosome occur about 8.7 times more than inter-chromosomal exchanges calculated on the basis of the DNA content of the chromosomes and random induction of aberrations in the total genome. Chromosomal organization in interphase nucleus appears to promote the formation of more intra-changes than inter-changes following X-irradiation, most probably due to close proximity of the two arms of a chromosome.

Micronuclei are formed during cell division either from lagging acentric fragments caused by chromosomal breakage or from whole chromosomes. Female Swiss mice were used to study the induction and persistence of micronuclei (MN) in isolated splenocytes up to 112 days after 2Gy whole-body X-irradiation. Micronucleus frequency in cytokinesis blocked binucleated cells was estimated using acridine orange staining. Fluorescence in situ hybridization (FISH) with murine minor satellite DNA probe was used to detect aneuploidy (MN with a centromere). The initial frequency of micronuclei immediately after X-ray exposure was found to be 39.6 per 100 cells. The MN frequency declined in an exponential manner during the subsequent period and at day 14, reached about half the value observed immediately after irradiation with a further 50% reduction by day 28 and only 3% MN could be detected at day 112 post-irradiation. Some induction of aneuploidy was observed after X-irradiation, deduced in the form of centromere-positive micronuclei (C + MN) as detected by the presence of a minor satellite signal after FISH. Of MN, 23% were centromere-positive in the first fixation time after X-ray exposure. Some of the MN showed two or more centromeric signals. The percent C + MN increased gradually until day 7 post-exposure. At day 14, this value started to decline, reaching 13% by 112 days post-irradiation. FISH with a biotinylated minor satellite probe can reliably be used in the cytokinesis blocked micronucleus assay to distinguish the clastogenic and/or aneugenic activity of a test chemical or physical agent.

In the present paper, the protective effect of beta-carotene was evaluated after whole body exposure of mice to 2 Gy of X-rays. Splenocytes, reticulocytes, bone marrow cells and spermatids were evaluated for the frequency of micronuclei (MN) induced by X-rays. Mice were treated (gavage) with beta-carotene (10, 25 and 50 mg/kg b.w.) for 5 consecutive days and, 4 h after the last treatment, the animals were irradiated. The results obtained showed different frequencies of X-ray-induced-MN between different cell populations analysed and also different response of these cells to the beta-carotene treatment. The radioprotective effect of beta-carotene was observed in splenocytes, reticulocytes, and spermatids but not in bone marrow cells. No dose-response relationship for beta-carotene was detected. The time of sampling, the sensitivity of the cells as well as the antioxidant activity of beta-carotene are discussed as important factors for the radioprotective action of this provitamin.

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.

X-ray induced chromosomal translocations were studied in mouse spermatogonial stem cells by meiotic analysis at the spermatocyte stage many cell generations after induction. Using a composite DNA probe for mouse chromosomes 1, 11 and 13, in combination with fluorescence in situ hybridization, the involvement of these three chromosomes in translocation formation was recorded. The obtained results at various sampling times ranging from 75 to 320 days after irradiation show no significant differences in the participation pattern of the painted chromosomes in multivalent formation with increasing sampling time. Pooling of the data at the different dose levels of 5 and 7 Gy indicated significant overrepresentation of the specifically stained bivalents in translocation formation. This suggests that clonal proliferation cannot be held responsible for the observed non-randomness. Experiments with the DNA-repair inhibitor 3-aminobenzamide showed no change in the recorded overrepresentation, indicating that it is probably not the repair of lesions that is causing this phenomenon but rather non-random induction.

Most mutagens and genotoxic carcinogens are efficient inducers of chromosomal alterations in exposed cells. Two important classes of aberrations, namely structural and numerical, are recognized and both types of aberrations are associated with congenital abnormalities and neoplasia in humans. These alterations can be easily detected and quantified in human peripheral blood lymphocytes. Conventional staining techniques can be used to detect these aberrations; this technique was used to estimate absorbed dose in the case of a radiation accident in Goiania, Brazil. A recently introduced fluorescent in situ hybridization technique (FISH) using DNA probes has increased the sensitivity and ease of detecting chromosome aberrations, especially stable chromosome aberrations. This technique allows, to some extent, the estimation of absorbed radiation dose from past exposures. Numerical aberrations can be directly estimated in metaphases by counting the number of FISH-painted chromosomes. Micronuclei are formed by lagging chromosome fragments or whole chromosomes during the anaphase stage of cell division. The nature of micronuclei as to whether they possess a centromere can be determined either by CREST staining (calcinosis, Raynoud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasia) or FISH with centromere-specific DNA probes. In several carcinogen-exposed populations, such as heavy smokers or people exposed to arsenic, aneuploidy appears to be more common than structural aberrations. In victims of radiation accidents, aneuploidy (hyperploidy) has been found to be common in addition to structural aberrations.

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 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.

Chromosomal aberrations in human lymphocytes were analyzed by fluorescence in situ hybridization (FISH) in the first 3 postirradiation (0 and 2 Gy) divisions. Cells were grown in the presence of BrdU, collected at different sampling times (47, 70 and 91 h) and analyzed using an alphoid centromeric probe and PCR amplified DNA libraries for chromosomes 2 and 8. Following differential staining of sister chromatids, the analyzed cells were identified to be either in the first, second or third mitosis after irradiation. The frequencies of both dicentrics and fragments showed a reduction of about 50% after each cell generation, whereas translocations were more persistent. Cells within the same postirradiation division showed higher aberration frequencies when derived from later sampling times, indicating a delay in progression of aberrant cells. As a result, the frequencies for dicentrics and fragments remained rather constant at different sampling times if the cell cycle parameter was not taken into account. Thus, the average generation time of the lymphocytes had a clear effect on the obtained aberration frequencies. The described method allows the study of the persistence of chromosome damage using the FISH technique during 3 subsequent cell divisions in vitro.

The efficiency of two methods of detection of translocations induced in mouse stem cell spermatogonia by X-ray doses of 2, 5 and 7 Gy was compared: classical multivalent analysis at diakinesis-metaphase I of meiosis and observation via fluorescence in situ hybridization analysis of mitotic or meiotic stages. Specific DNA libraries for chromosomes 1, 11 and 13 were used. The results obtained indicate that (a) chromosomes 1, 11 and 13 are more involved in multivalent formation than expected on the basis of DNA content and (b) if the mitotic FISH analysis data are corrected for the observed over-representation, the frequencies of induced translocations are similar to those recorded in the classical multivalent studies, suggesting equal scoring efficiencies in both systems.

Radiation induced chromosomal aberrations in bone marrow cells of scid and normal mice were studied at different sampling times. Fluorescence in situ hybridization (FISH) with DNA libraries specific for chromosomes 1, 11 and 13 was applied to identify the stable types of chromosomal aberrations in addition to the unstable ones. The results obtained confirm earlier observations on stem cell spermatogonia in that, contrary to the situation in normal mice, only very low levels of translocations could be recovered from scid mice at relatively long sampling times (3 weeks). However, studies at a 24 h sampling period demonstrated substantial induction of translocations in scid mice. This suggests enhanced elimination of translocation carrying cells in scid mice during successive cell proliferation, possibly via falling apart of the translocation at the original points of exchange or due to lethal damage at the translocation break points.

Most of the recently developed tests for detecting aneugenic activity of chemicals are based on the induction of micronuclei (MN) in cytokinesis-blocked (CB) binucleated cells. In such a test, aneugens can be discriminated from clastogens by checking for the presence of centromeres in the MN, indicating the loss of whole chromosomes. Tracing particular chromosomes in interphase nuclei using fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes is another method used for detecting numerical chromosome aberrations. Here, we describe a method using a cytokinesis-blocked MN assay in combination with identifying specific chromosomes of mice. For this purpose transgenic mice with foreign DNA inserted in three pairs of their chromosomes were generated. Splenocytes of these mice were cultured and treated in vitro with vinblastine (VBL) or X-rays, followed by recovery in medium containing cytochalasin B. By tracing the marker chromosomes in binucleated splenocytes, reciprocal products of chromosome malsegregation to the daughter nuclei could be easily traced. The results showed that besides clastogenic activity, X-rays also exhibited aneugenic activity. Treatment with vinblastine showed a close relationship between micronuclei induction and chromosome malsegregation, although at higher doses malsegregation processes became more prominent. Simultaneous malsegregation of more than one chromosome was observed frequently, but the three marker chromosomes were found to be randomly involved in this process.

Ionizing radiation and restriction endonucleases are very efficient in inducing chromosomal aberrations (CAs). These aberrations are mainly consequences of misrepair of DNA double-strand breaks (DSBs). The fast repairing component of DSBs induced by ionizing radiation seems to be responsible for exchange aberration. Use of premature chromosome condensation technique in combination with DNA repair inhibitors such as ara A has given valuable information on the assessment of the frequencies of initial chromosome breaks and the kinetics of their repair following low LET radiation. The recently developed 'chromosome painting' technique using chromosome-specific libraries has also increased considerably the resolution of identifying and scoring of CAs. After low LET radiation, stable chromosome exchanges (translocations) are induced more frequently than unstable chromosome exchanges (dicentrics). Fluorescence in situ hybridization employing telomeric probe has made it possible to score efficiently exchange aberrations involving the acrocentric chromosomes of mouse. Chinese hamster cells have several intercalary telomeric sequences present in most of the chromosomes. These telomeric blocks have been found to be associated with chromosomal aberrations induced by restriction endonucleases and short wave UV and evidence has been obtained for apparent amplification of telomeric sequences at the break points.

We describe here the development of mouse chromosome-specific DNA libraries and their use in the detection of radiation-induced chromosome aberrations by fluorescence in situ hybridization. Large metacentric chromosomes, resulting from a translocation involving chromosomes 1, 11 and 13, were flow-sorted. Using a slit-scan technique for morphometric analysis, metacentric chromosomes were separated from normal acrocentric chromosomes and their aggregates. DNA from the metacentric chromosomes was amplified by PCR using the linker/adaptor method. In this pilot study, mouse was whole-body irradiated with 1, 2 and 3 Gy and aberrations were scored in metaphase spreads of splenocytes cultured in vitro. The results indicate that directly after radiation exposure, stable and unstable aberrations are induced at about equal frequencies in the splenocytes. The availability of chromosome-specific probes for mouse may prove very useful when analysing the behaviour of stable aberrations, as well as the testing of many suspected mutagenic carcinogens and aneugens in vivo for induction of chromosomal translocations and non-disjunction, respectively.