Radiation-induced Chromosome Aberrations Research Articles

Radioadaptation in Indian muntjac fibroblast cells induced by low intensity laser irradiation

Earlier reports have indicated that an adaptive, protective response to ionizing radiation is inducible by pre-treatment with low intensity laser irradiation (LILI). We have investigated the potential of LILI to induce an adaptive response against the damaging effects of ionizing radiation in Indian muntjac fibroblasts. LILI at 660, but not 820 nm, at 11.5 and 23.0 J/cm2, induced an apparent adaptive response in the form of a reduction in the frequency of radiation-induced chromosome aberrations, but not in cell survival. There was also a trend towards a reduction in the level of single-stranded and double-stranded DNA breaks induced by ionizing radiation when cells were preconditioned with LILI. However, this did not contribute to the reduced chromosome aberration frequency. Further analysis revealed that the reduced aberration frequency was caused by a laser-induced extension of G2 delay. The adaptive response was therefore the result of cell cycle modulation by LILI, at a wavelength where there is no known DNA damaging effect to induce the checkpoint mechanisms that are normally responsible for altering cell cycle progression.

Influence of sodium butyrate on the induction of radiation-induced chromosomal aberrations and sister chromatid exchanges in Chinese hamster ovary (CHO) cells

CHO cells were pre-treated with sodium butyrate (SB) for 24 h and then X-irradiated in G1. Metaphases were scored for the induction of chromosomal aberrations and sister chromatid exchanges (SCEs). The data were compared with those obtained after irradiation of cells not pre-treated with SB and showed that SB has different effects on the endpoints examined. The frequencies of dicentric chromosomes were elevated and of small acentric rings (double minutes, DMs) reduced. These results are discussed to be a consequence of conformational changes in hyperacetylated chromatin which could lead to more interchromosomal and to less intrachromosomal exchanges. SB itself induces a few SCEs but suppresses the induction of SCEs by X-rays. We assume that a minor part of radiation induced SCEs are `false' resulting from structural chromosomal aberrations, such as inversions, induced in G1. Inversions are the symmetrical counterparts of DMs. If inversions are suppressed by SB treatment to a similar extent as DMs a small reduction of SCEs by SB can be expected.

Radiation induced chromosome aberrations and clonogenic survival in human lymphoblastoid cell lines with different p53 status.

To better understand the relation of radiation induced chromosome aberrations and clonogenic survival in cells with different p53 status. The human lymphoblasts TK6 and WTK1 were derived from the same donor, but differ in radiosensitivity, p53 status and kinetics of apoptosis. TK6 cells have wild type p53 (p53wt), whereas WTK1 cells have a mutated, non-functional p53 (p53mut). Additionally, a HPV16 E6 transfected TK6 cell line (TK6E6), which is also negative for p53 function (p53neg), was studied. The cells were irradiated, incubated with colcemid, hypotonically lysed and fixed. After staining with Giemsa, asymmetric chromosomal exchange type aberrations were counted in 50 mitoses each per dose point (0 to 4 Gy). Clonogenic survival was determined using the microtiter plate assay. All experiments were performed in triplicate. WTK1 (p53mut) show a higher spontaneous frequency of chromosome aberrations than TK6 (p53wt). No significant differences were noted in radiation induced aberration frequency. TK6E6 (p53neg) show comparable aberration frequencies like TK6. However, the dose required to reduce survival to 10% (D10) was about 2 Gy for TK6 and TK6E6, whereas the D10 for WTK1 was approximately 3 Gy. The p53 status influences the radiosensitivity in this lymphoblast cell system showing a high rate of radiation induced apoptosis. Cells with p53mut (WTK1), survive with a damaged genome, because they do not undergo apoptosis to loose their clonogenicity. There was no difference between the p53wt (TK6) and p53neg cells (TK6E6) suggesting a suppression of radiation induced apoptosis by p53mut.

Low-dose whole-body irradiation induces alteration of protein expression in mouse splenocytes

In previous studies, the stimulatory effect on immunological function and adaptive response in splenocytes were found after exposure of mice to low-dose whole-body irradiation (LD-WBI). Protein synthesis was found to be required for these responses. The present study, therefore, attempts to investigate the protein changes in mouse splenocytes after exposure to LD-WBI. Two-dimensional gel electrophoresis was used to show the alterations of proteins in nuclei, cytoplasm and extracellular fluid of the splenocytes at 4 h after mice exposed to 75 mGy X-rays. As compared to control, changed expressions of 20, 15 and 6 proteins, in sizes ranging from 10 to 69 kDa with p I of 5.0–8.2, were found in the nuclei, cytoplasm and extracellular fluid of splenocytes, respectively. One protein, shown in the cytoplasm of splenocytes of control mice, appeared in extracellular fluid of splenocytes after LD-WBI. Two proteins, shown in cytoplasm, and one protein, shown in extracellular fluid of splenocytes in control mice, appeared in the nuclei after LD-WBI. Time-course of protein synthesis varied in different proteins after LD-WBI. These results suggest that alterations of protein expressions and redistribution of proteins between intracellular and extracellular compartments occurred in the splenocytes after LD-WBI. Protein extract from LD-WBI splenocytes was fractionated by Sephadex G-100. Fractions 61–80 contained proteins able to protect lymphocytes in vitro from radiation-induced chromosome aberrations. These findings are of importance in elucidating mechanisms of immuno-enhancement and adaptive response induced by low-dose radiation, although the features and the functions of these proteins remain to be elucidated in future studies.

The contribution of DNA ploidy to radiation sensitivity in human tumour cell lines.

The contribution of DNA ploidy to radiation sensitivity was investigated in a group of eight human tumour cell lines. As previous studies suggest, while more aneuploid tumours tend to be more radioresistant, there is no significant relationship between ploidy and radiation sensitivity (SF2). The failure to observe a significant effect of ploidy on radiation sensitivity is due to the complex and multifactorial basis of radiation sensitivity. When we determined the relationship between survival and radiation-induced chromosome aberration frequency, a measure independent of most other modifiers of sensitivity, we observed a direct relationship between ploidy and mean lethal aberration frequency. The mean lethal frequency of aberrations increased from about 1 for diploid cells to about 2 for tetraploid cells. The mean lethal frequency of aberrations was independent of DNA repair variations. These observations demonstrate that changes in DNA ploidy are an important contributor to radiation sensitivity variations in human tumour cell lines. Therefore, any battery of predictive assays should include DNA ploidy measurements. © 1999 Cancer Research Campaign

Chk1 complements the G2/M checkpoint defect and radiosensitivity of ataxia-telangiectasia cells.

Cells from patients with the human genetic disorder ataxia-telangiectasia (A-T) are defective in the activation of cell cycle checkpoints in response to ionizing radiation damage. In order to understand the role of ATM in checkpoint control we investigated whether Schizosaccaromyces pombe chk1, a protein kinase implicated in controlling the G2 DNA damage checkpoint, might alter the radiosensitive phenotype in A-T cells. The fission yeast chkl gene was cloned into an EBV-based vector under the control of a metallothionein promoter and transfected into A-T lymphoblastoid cells. Induction of chk1 enhanced the survival of an A-T cell line in response to radiation exposure as determined by cell viability and reduction of radiation-induced chromosome aberrations. This can be accounted for at least in part by the restoration of the G2 checkpoint to chk1 expressing cells. There was no evidence that chk1 expression corrected either the G1/S checkpoint or radioresistant DNA synthesis in S phase in these cells. These results suggest that chk1 when overexpressed acts downstream from ATM to restore the G2 checkpoint in these cells and correct the radiosensitive phenotype. These data allow us to dissociate individual checkpoint events and relate them to the radiosensitive phenotype in A-T cells.

Potential of radiation-induced chromosome aberrations to predict radiosensitivity in human tumour cells

Purpose: To validate whether the number of aberrations could be used as a measure of the radiosensitivity of human tumour cells. If so, this would potentially provide a more rapid method than the colony assay to predict radiocurability in human tumour biopsy material. Materials and methods: A panel of 13 human tumour cell lines was investigated, covering a wide range of radiosensitivities. Fluorescence in situ hybridization (FISH) employing whole chromosome probes was used to detect aberrations. Results: A dose-dependent increase in radiation-induced chromosome aberrations was observed in all cell lines. A good correlation (r=0.90) was found between cell survival and total chromosome aberrations in 12 of the 13 cell lines (92%), with one exception. A poorer correlation was observed between cell survival and stable- (r=0.85) and unstable-type aberrations (r=0.81). Survival-aberration correlations for individual radiation doses were worse, although statistically significant. The exceptional cell line showed significantly more aberrations for a given level of cell kill than expected based on data for the other lines. Conclusion: This study indicates that radiation-induced chromosome aberrations can be used as a potential predictor of intrinsic radiosensitivity for the majority of human tumours when more than one dose level is tested. This could aid the design of radiotherapy schedules for each individual patient, or in the decision of whether to use an alternative therapy.

Potentiation by turmeric and curcumin of gamma-radiation-induced chromosome aberrations in Chinese hamster ovary cells.

The effect of turmeric and curcumin, two natural antioxidants, on the frequencies of chromosome aberrations induced in Chinese hamster ovary (CHO) cells by gamma-radiation was investigated. Cells were treated with three concentrations of each drug, turmeric (100, 250, and 500 microg/ml) and curcumin (2.5, 5, and 10 microg/ml), and then irradiated (2.5 Gy) during different phases of the cell cycle. Turmeric was not clastogenic by itself, whereas curcumin at 10 microg/ml enhanced the chromosomal damage frequency. Neither of the two antioxidants showed protective effect against the clastogenicity of gamma-radiation. Instead, an obvious increase in the frequencies of chromosome aberrations was observed when turmeric at 500 microg/ml was associated with gamma-radiation during G2/S phase, and curcumin at 10 microg/ml plus gamma-radiation during S and G2/S phases of the cell cycle. The results clearly indicate the exacerbated effect of turmeric and curcumin on radiation-induced clastogenicity, suggesting that these antioxidants are also potentiating agents depending on the experimental conditions.

Effect of estradiol on radiation-induced chromosome aberrations in human lymphocytes.

As a part of studies on physiological factors that affect radiosensitivity, we examined the in vitro effect of estradiol (E2) on the yield of radiation-induced chromosome aberrations in human peripheral lymphocytes. Lymphocytes were cultured for 3 days in the medium containing E2 at 0-100,000 ng/ml. On the second day, they were irradiated by X-rays at 3 Gy, and then 2% phytohemagglutinin and 0.05 microgram/ml colcemid were added to the medium. After further 48 h, mitotic indices and the yields of chromosome aberrations were examined at various E2 concentrations. E2 treatment at concentrations above 1000 ng/ml resulted in dose-related inhibition of mitosis. Repeated experiments showed that the yield of dicentrics plus centric rings in the culture containing E2 at 100 ng/ml was significantly higher than the yields at 0 ng/ml. Similarly, the yield of total chromosome breaks in the culture containing E2 at 100 ng/ml was significantly higher than that at 1 ng/ml. This study provides the direct evidence in human that radiosensitivity may vary in relation to hormonal conditions.

Radiation-induced chromosomal aberrations and haematological alterations in hospital workers.

Long-term exposure to low doses of ionizing radiation may affect cells and tissues and result in various adverse health effects. The purpose of this study was to investigate whether chromosome aberrations and haematological alterations could be used as biomarkers of possible radiation injury in workers exposed to ionizing radiation. Groups totalling 323 medical professionals handling X-ray equipment and 160 control subjects were examined for incidence of chromosome aberrations and changes in leukocyte, lymphocyte and thrombocyte counts. The incidence of all types of chromosome aberrations was higher in the exposed groups than in controls, yet no significant difference was found between the exposed groups. A many-fold increase in chromosome aberration frequency in all exposed groups was not followed by a corresponding haematological depression. This suggests that chromosome aberrations are a significantly more sensitive indicator of changes caused by low doses of ionizing radiation than haematological alterations.

Multicolour FISH painting for the analysis of chromosomal aberrations induced by 220kV X-rays and fission neutrons

Purpose : The quantification of radiation-induced chromosome aberrations identified by multicoloured FISH painting and classified according to diff erent nomenclature systems (PAINT, S&S and a conventional method). Material and methods : Blood samples were irradiated with five diff erent doses of 220kV X-rays or fission neutrons respectively. Cell cycle-controlled, multicoloured FISH painting was performed with a cocktail of chromosomes 1, 4, 12 and a pancentromeric probe. Results : Ten aberration parameters or categories were selected according to the three nomenclature systems and dose-response curves were constructed for the observed yields. Fitted coeffi cients of the linear-quadratic dose-response function show the relative importance of the quadratic term for aberration parameters of the low-LET radiation (X-rays) data set and of the linear term for those of the high-LET radiation (fission neutrons) data set. The relative proportion of complex aberrations observed was larger for the densely ionizing fission neutrons than for sparsely ionizing X-rays. Conclusion : Compared with single-colour FISH painting, a multicolour approach provides extended information for a mechanistic and quantitative interpretation of radiation-induced chromosome aberrations. The choice of a nomenclature system and the selection of an appropriate aberration parameter or category depend on these specific aspects. Practical application requires a rapid and reproducible description of the observed painting patterns and should also throw light on the origin of aberrations.

Modulation of the clastogenic activity of gamma-irradiation in buthionine sulphoximine-mediated glutathione depleted mammalian cells

Purpose: Chromosome aberrations (CA) were used as an endpoint to investigate the effect of buthionine sulphoximine (BSO), a potent glutathione-depleting agent, on the radiosensitivity of mammalian cells. The aim was to obtain information about the role of glutathione (GSH) in physicochemical and biochemical processes in irradiated cells. Materials and methods: CA were scored from first cycle metaphases in irradiated BSO-pretreated and untreated samples. BSO exposure was for 10h in mouse bone marrow cells in vivo and 5h for human lymphocytes in vitro. In further experiments fresh blood was irradiated on ice and immediately after irradiation GSH/GSH-ester was added. Results: In both the systems BSO-treated samples showed higher sensitivity to radiation than BSO untreated samples. The frequency of all types of CA increased except exchange aberrations. GSH/GSH-ester treatment given after irradiating the cells at 4 C reduced the frequency of deletions and increased the frequency of exchange aberrations. Conclusions: Data indicate that BSO-mediated GSH depletion increased radiation-induced chromosome aberrations, apart from exchange aberrations. This could be due to reduction in the free-radical scavenging effect of GSH, a failure in rejoining of DNA double-strand breaks, or induction of apoptosis.

Delayed cell death, giant cell formation and chromosome instability induced by X-irradiation in human embryo cells.

We studied X-ray-induced delayed cell death, delayed giant cell formation and delayed chromosome aberrations in normal human embryo cells to explore the relationship between initial radiation damage and delayed effect appeared at 14 to 55 population doubling numbers (PDNs) after X-irradiation. The delayed effect was induced in the progeny of X-ray survivors in a dose-dependent manner and recovered with increasing PDNs after X-irradiation. Delayed plating for 24 h post-irradiation reduced both acute and delayed lethal damage, suggesting that potentially lethal damage repair (PLDR) can be effective for relieving the delayed cell death. The chromosome analysis revealed that most of the dicentrics (more than 90%) observed in the progeny of X-ray survivors were not accompanied with fragments, in contrast with those observed in the first mitosis after X-irradiation. The present results indicate that the potentiality of genetic instability is determined during the repair process of initial radiation damage and suggest that the mechanism for formation of delayed chromosome aberrations by radiation might be different from that of direct radiation-induced chromosome aberrations.

Generalized concept of the LET-RBE relationship of radiation-induced chromosome aberration and cell death.

The frequency of chromosome aberrations per traversal of a nucleus by a charged particle at the low dose limit increases proportionally to the square of the linear energy transfer (LET), peaks at about 100 keV/micron and then decreases with further increase of LET. This has long been interpreted as an excessive energy deposition over the necessary energy required to produce a biologically effective event. Here, we present an alternative interpretation. Cell traversed by a charged particle has certain probability to receive lethal damage leading to direct death. Such events may increase with an increase of LET and the number of charged particles traversing the cell. Assuming that the lethal damage is distributed according to a Poisson distribution, the probability that a cell has no such damage is expressed by e-cLx, where c is a constant, L is LET, and x is the number of charged particles traversing the cell. From these assumptions, the frequency of chromosome aberration in surviving cells can be described by Y = alpha SD + beta S2D2 with the empirical relation Y = alpha D + beta D2 in the low LET region, where S = e-cL, alpha is a value proportional to LET, beta is a constant, and D is the absorbed dose. This model readily explains the empirically established relationship between LET and relative biological effectiveness (RBE). The model can also be applied to clonogenic survival. If cells can survive and they have neither unstable chromosome aberrations nor other lethal damage, the LET-RBE relationship for clonogenic survival forms a humped curve. The relationship between LET and inactivation cross-section becomes proportional to the square of LET in the low LET region when the frequency of a directly lethal events is sufficiently smaller than unity, and the inactivation cross-section saturates to the cell nucleus cross-sectional area with an increase in LET in the high LET region.

Reduction of radiation-induced chromosome aberration and apoptosis by dithiothreitol.

We have examined in vitro and in vivo radioprotective effects of a well-known thiol-containing compound, dithiothreitol (DTT). The treatment of both 0.5 and 1 mM of DTT significantly increased clonogenic survival of gamma-ray irradiated Chinese hamster (V79-4) cells. In order to investigate the possible radioprotective mechanism of DTT, we measured gamma-ray induced chromosome aberration by micronucleus assay. In the presence of 0.5 mM or 1 mM DTT, the frequencies of micronuclei were greatly reduced in all dose range examined (1.5-8 Gy). Slightly higher reduction in micronucleus formation was observed in 1 mM DTT-treated cells than in 0.5 mM DTT-treated cells. In addition, incubation with both 0.5 and 1 mM of DTT prior to gamma-ray irradiation reduced nucleosomal DNA fragmentation at about same extent, this result suggests that treatment of DTT at concentrations of 0.5 and 1 mM reduced radiation-induced apoptosis. In vivo experiments, we also observed that DTT treatment reduced the incidence of apoptotic cells in mouse small intestine crypts. In irradiated control group 4.4 +/- 0.5 apoptotic cells per crypt were observed. In DTT-administered and irradiated mice, only 2.1 +/- 0.4 apoptotic cells per crypt was observed. In vitro and in vivo data obtained in this study showed that DTT reduced radiation-induced damages and it seems that the possible radioprotective mechanisms of action of DTT are prevention of chromosome aberration.

Radiation induced chromosome aberrations: some biophysical considerations

The implications of recent results using FISH chromosome painting and soft X-ray exposures for the mechanisms of chromosome aberration formation are discussed. It is concluded that the evidence in favour of exchange aberrations arising from one radiation induced chromosome break has increased to the point where a ‘change in paradigm’ from the older breakage-reunion hypothesis needs to be taken seriously into account. A potential role for recombinational repair of DNA double strand breaks, as known in yeast, in the formation of aberrations in mammalian cells is presented and the relationship between DNA repair studies and radiation cytology is emphasized.

The F Value Cannot Be Ruled out as a Chromosomal Fingerprint of Radiation Quality

The F value, the ratio of inter- to intrachromosomal interchanges, as a biomarker for densely ionizing radiation which was proposed by Brenner and Sachs (Radiat. Res. 140, 134-142, 1994) has been a matter of repeated discussion. We examined our experimental data on radiation-induced chromosome aberrations in human peripheral blood lymphocytes for the F value as measured by the ratio of dicentrics to centric rings. Because of the rarity of aberrations, the F value showed a considerable variability, with a large error range particularly in the low-dose range of low-LET radiation. However, the data showed a general trend that the F value tended to be lower for high-LET than low-LET radiations. The differential F value was more pronounced at low doses and diminished with increasing dose; the F values of all radiations tended to converge toward a similar value at high doses. The limiting F value at the lowest doses, or the F0 value, was dependent on LET for high-energy radiations that can produce an array of DNA double-strand breaks along the track of the charged particle. However, LET and dose dependence were not seen for the low-energy photons, where spatially uncorrelated random breaks were produced by independent photoabsorption events.

Clinical radiohypersensitivity screening using radiation-induced chromosomal aberrations.

Severe acute toxicity to radiotherapy (radiohypersensitivity) can limit the effective use of radiotherapy and it is not usually possible to identify such individuals before treatment commences. Five patients with acute radiohypersensitivity (RH) were detected over a 3-year period. All five RH subjects demonstrated a significantly higher degree of radiation-induced chromosomal aberrations (ICA) in fresh blood lymphocytes when compared to normal controls. Results indicate the feasibility of using the ICA assay in conjunction with other tests to screen for radiosensitivity.

Absence of ATM truncations in patients with severe acute radiation reactions

Purpose: Severe acute toxicity limits the effective use of radiotherapy in patients who are radiosensitive, and it is not usually possible to identify these radiohypersensitive (R-H) individuals before treatment commences. Five such R-H patients were detected over a 3-year period. We undertook this study to determine whether the severe acute radiohypersensitivity of these five individuals showed any correlation with cellular and molecular parameters known to be abnormal in radiosensitivity-related syndromes such as ataxia–telangiectasia (A-T). Methods and Materials: Lymphoblastoid cells were isolated from fresh blood from the 5 R-H individuals who had previously demonstrated clinical R-H at least 9 months prior to sampling. Lymphoblastoid cell lines (LCLs) were established to determine the extent of postradiation chromosomal aberrations, cell cycle delay, cell proliferation, and tumor suppressor p53 protein stabilization. The polymerase chain reaction (PCR) and protein truncation (PTT) assays were used to test for the possibility of mutations in the gene mutated in A-T, termed ATM. Results: LCLs derived from R-H subjects retained a significantly higher degree of radiation-induced chromosomal aberrations when compared to normal control LCLs. p53 stabilization by ionizing radiation appeared normal in all but one R-H subject. There was no evidence of A-T gene truncation mutations in any of the R-H subjects tested. Conclusions: All R-H subjects in this study had their cellular radiosensitivity confirmed by the chromosomal aberration assay. Delayed p53 stabilization at 4 hours postirradiation in one R-H subject suggested that different etiologies may apply in the radiohypersensitivity investigated in this study.

Radiation-induced chromosome aberrations in Saccharomyces cerevisiae: influence of DNA repair pathways.

Radiation-induced chromosome aberrations, particularly exchange-type aberrations, are thought to result from misrepair of DNA double-strand breaks. The relationship between individual pathways of break repair and aberration formation is not clear. By electrophoretic karyotyping of single-cell clones derived from irradiated cells, we have analyzed the induction of stable aberrations in haploid yeast cells mutated for the RAD52 gene, the RAD54 gene, the HDF1(= YKU70) gene, or combinations thereof. We found low and comparable frequencies of aberrational events in wildtype and hdf1 mutants, and assume that in these strains most of the survivors descended from cells that were in G2 phase during irradiation and therefore able to repair breaks by homologous recombination between sister chromatids. In the rad52 and the rad54 strains, enhanced formation of aberrations, mostly exchange-type aberrations, was detected, demonstrating the misrepair activity of a rejoining mechanism other than homologous recombination. No aberration was found in the rad52 hdf1 double mutant, and the frequency in the rad54 hdf1 mutant was very low. Hence, misrepair resulting in exchange-type aberrations depends largely on the presence of Hdf1, a component of the nonhomologous end-joining pathway in yeast.