mFISH analysis of chromosome aberrations in workers occupationally exposed to mixed radiation

We performed a study on the presence of chromosome aberrations in a cohort of plutonium workers of the Mayak production association (PA) with a mean age of 73.3 ± 7.2 years to see whether by multi-color fluorescence in situ hybridization (mFISH) translocation analysis can discriminate individuals who underwent occupational exposure with internal and/or external exposure to ionizing radiation 40 years ago. All Mayak PA workers were occupationally exposed to chronic internal alpha-radiation due to incorporated plutonium-239 and/or to external gamma-rays. First, we obtained the translocation yield in control individuals by mFISH to chromosome spreads of age-matched individuals and obtained background values that are similar to previously published values of an international study (Sigurdson et al. in Mutat Res 652:112–121, 2008). Workers who had absorbed a total dose of >0.5 Gy external gamma-rays to the red bone marrow (RBM) displayed a significantly higher frequency of stable chromosome aberrations relative to a group of workers exposed to <0.5 Gy gamma-rays total absorbed RBM dose. Thus, the translocation frequency may be considered to be a biological marker of external radiation exposure even years after the exposure. In a group of workers who were internally exposed and had incorporated plutonium-239 at a body burden >1.48 kBq, mFISH revealed a considerable number of cells with complex chromosomal rearrangements. Linear associations were observed for translocation yield with the absorbed RBM dose from external gamma-rays as well as for complex chromosomal rearrangements with the plutonium-239 body burden.     

Quantitative analyses of the induction of chromosome aberrations and sister-chromatid exchanges in human lymphocytes exposed to gamma-rays and mitomycin-C in combination

Most chemicals are S-dependent and are potent inducers of SCE, but do not produce chromosome-type aberrations in the first metaphases after exposure. Ionizing radiation, which is an S-independent agent, produces chromosome-type aberrations, especially dicentrics and rings, but inefficiently produces chromatid-type aberrations. A series of experiments has been performed to investigate whether cytogenetic damage induced by ionizing radiation (gamma-rays) might be assessed separately from that induced by the alkylating chemical, mitomycin C (MMC), when human lymphocytes were exposed to these 2 agents in combination. Whole-blood cultures of human lymphocytes in G0 phase were exposed to gamma-rays and MMC in combination or separately. Cytogenetic analyses were done for both chromosome aberrations (CA), analyzed in cultures incubated for 56 h without BrdUrd, and sister-chromatid exchanges (SCEs) in cultures incubated for 72 h with BrdUrd. The frequency of chromosome-type aberrations (dicentrics and rings) increased with increasing doses of gamma-rays from 0.5 to 4.0 Gy. The dose-response relationships were the same with or without concomitant treatment with MMC (10(-6) M). Although the SCE frequency increased with increasing doses of MMC, the increase was nearly the same as when cells were treated with both MMC and gamma-rays (2 Gy). There was no interaction between MMC and gamma-rays concerning these 2 endpoints.     

Cellular reactions induced by chronic exposure to 14C beta-rays

In V-79 cells the Effects of chronic exposure on induction of chromosome aberrations and abnormal metaphases as well as on efficiency of subsequent exposure to 2 Gy gamma-rays were investigated. It was found that chronic exposure increased the yield of chromosome aberrations as well as abnormal metaphases (spread-metaphases and apoptotic metaphases). In spite of the level of damages in cells, the chronic beta-exposure protected cells against the additional induction of chromosomal aberrations by subsequent exposure to higher acute dose of gamma-irradiation. Cytogenetic adaptive response was retained in the surviving progeny of irradiated cells which were cultured in free medium during 40, 70 days or one year after chronic exposure. At this time the level of residual chromosome aberrations, colony forming ability and distribution of the cells by the number of chromosomes were almost the same as in unirradiated cells. However, the high level of abnormal metaphases and half as much of cells in colony in the surviving progeny of irradiated cells in comparison with unirradiated cell, allow us to suggest that the chronic exposure induced the selection of adaptive forms in condition of the higher level of radiation.     

The repair of DNA damage induced in human lymphocytes by gamma rays and fast neurons

When Go human lymphocytes are exposed either to gamma-rays or to d(50)-Be neutrons and then immediately incubated in presence of cytosine arabinoside, the frequency of chromosomal aberrations which is normally observed after radiation exposure only is sharply increased. This enhancement of the aberrations, particularly the dicentrics, is, however, less marked when cytosine arabinoside is administered at longer intervals of time after irradiation. For gamma-rays, the treatment with cytosine arabinoside has no effect on the dicentrics yield when given 5 h after irradiation, indicating that the repair is completed within the 5 h after irradiation and that the lesions are not anymore available to produce exchange aberrations. For d(50)-Be neutrons, the time of repair takes approximately 5 h after a dose of 2.0 Gy, whereas it appears to be shorter (3 h) after a dose of 0.5 Gy.     

Chromosomal hypersensitivity in mutant MCN-151 mouse cells exposed to mitomycin C

After exposure to mitomycin C, the mouse lymphoma cell mutant MCN-151, previously shown to be sensitive to the toxic effect of the drug, demonstrates higher frequencies of chromatid-type aberrations and of aberrant cells in comparison to L5178Y cells. At any given dose of mitomycin C, the frequencies of chromosomal aberrations increased with time reaching a plateau in both cell strains, with the mutant in all cases being more sensitive than normal cells.     

Radioprotection by caffeine pre- and post-treatment in the bone marrow chromosomes of mice given whole-body gamma-irradiation.

The effect of caffeine given as pre- and post-treatment in mice exposed to whole-body gamma-irradiation (1.5 Gy 60Co gamma-rays) was studied. The pre-treatment was either acute or chronic. The acute dose (5 mg/kg and 15 mg/kg body weight) was in the form of an injection given intraperitoneally, 30 min before irradiation. The chronic administration was in the form of caffeine solution (4.208 x 10(-3) M and 7.72 x 10(-4) M) contained in the drinking water that mice had had ad libitum access to instead of plain drinking water for 5 weeks prior to radiation exposure. The acute pre-treatment with caffeine reduced the radiation-induced frequency of chromosomal aberrations discernibly, whereas the chronic pre-treatment afforded a much more significant degree of radioprotection. The caffeine post-treatment (5 mg/kg and 15 mg body weight) was given in the form of an intraperitoneal injection to the mice immediately following whole-body gamma-irradiation. It is noted that both post-treatment concentrations of caffeine also significantly reduced the frequency of chromosomal aberrations induced by gamma-rays. These data are briefly discussed in terms of possible mechanistic considerations.     

Anticlastogenic effects of a polyvitamin product, 'Pharmavit', on gamma-ray induction of somatic and germ cell chromosome aberrations in the mouse.

The polyvitamin product 'Pharmavit' (Pv), comprising vitamins A, D2, B1, B2, B6, C, E, nicotinamide, and calcium pantothene, was tested for anticlastogenic properties against gamma-rays in mice. Pretreatment with Pv consisted of daily administration by gavage for 30 days at dose levels corresponding to clinical recommendations for an adult human, as recalculated in terms of mg/kg. Findings indicated a reduction of chromosome aberrations in bone marrow cells from mice exposed to 3.0 Gy 137Cs gamma-rays; the reduction concerned predominantly fragments of the chromatid type. Furthermore, a reduction factor of 1.6 was obtained for the frequency of reciprocal translocations induced by spermatogonial irradiation in mice exposed to 4.0 Gy gamma-rays. Pretreatment with vitamin C alone, at the dose present in Pv, proved nearly ineffective in protecting from chromosome aberrations in bone marrow cells. Pharmavit is believed to be a promising agent for application to human populations exposed to the carcinogenic and genetic hazards of ionizing radiation.     

Study on the repair of the radioinduced lesions involved in the formation of chromosomal aberrations in G0 human lymphocytes after exposure to gamma-rays and fast neutrons

Cytosine arabinoside (ara-C), an inhibitor of DNA synthesis and repair, has been used to study the mechanisms of formation of chromosomal aberrations after exposure to low- and high-LET radiation. When G0 human lymphocytes were exposed either to gamma-rays or to d(50 MeV)-Be neutrons and immediately treated with ara-C for increasing periods of time, the frequency of aberrations (dicentrics) increased sharply. For gamma-rays, the enhancement increased with the duration of the treatment up to 5 h, whereas for neutrons, an ara-C treatment lasting for 5 h was no more effective than treatment for 3 h. These results were confirmed by the second experiment in which ara-C was administered for 3 h with an increasing time delay following irradiation. Since no increase in the dicentric frequency was observed when ara-C was administered 5 h after gamma-irradiation, it is suggested that the induced breaks rejoined within that time. For neutrons, the data were conflicting since the repair was completed within 3 h after a dose of 0.5 Gy, and in approximately 5 h after a dose of 2.0 Gy. From both experiments, it appears that gamma-rays and fast neutrons produce similar types of lesions, as ara-C increased the frequencies of aberrations induced by both types of radiation. However, the ara-C treatment resulted in a smaller increase in aberrations following neutron irradiation. According to the enzymatic nature of break formation and the mode of action of ara-C on the polymerase activity, it is suggested that, in addition to double-strand breaks, single-strand breaks could be the lesions involved in the repair processes inhibited by ara-C. Single-strand breaks formed directly or by secondary reactions would, therefore, be one of the major lesions responsible for the aberrations produced by gamma and neutron radiations.     

Structural genomic damages in workers of plutonium production

The research objective is assessment of structural genomic damages in plutonium workers. The study group included the Mayak nuclear workers subject to chronic occupational exposure to incorporated 239Pu and/or external gamma-rays. The analysis was performed based on the culture of lymphocytes in peripheral blood. The yield of intra-chromosomal exchange aberrations of chromosomal type on stained slides was analyzed using in situ fluorescent hybridization, mBAND. Linear relationships were revealed between (a) the total yield of chromosomal type aberrations (e.g. intra- and inter-chromosomal ones) and an absorbed dose from external exposure of the red bone marrow to gamma-rays, an absorbed dose from internal exposure to a-radiation from incorporated 239Pu; and (b) the yield of intra-chromosomal exchange aberrations of chromosomal type and an absorbed dose from exposure of the red bone marrow to 239Pu and 239Pu body burden.     

Radio-adaptive response: characterization of a cytogenetic repair induced by low-level ionizing radiation in cultured Chinese hamster cells

Pretreatment with low doses of beta-rays from incorporated tritiated thymidine ([3H]dThd) or of Co-60 gamma-rays (1 or 5 cGy) rendered actively growing Chinese hamster V79 cells more resistant to the induction of micronuclei or sister-chromatid exchanges (SCEs) by a subsequent high dose of gamma-rays (1 Gy). This adaptive response to ionizing radiation (radio-adaptive response) can be induced by an optimal range of low doses of 3H beta-rays, but not by much lower or higher adapting doses. Full expression of the adaptive response induced by the exposure to low doses of 60Co gamma-rays occurred 4 h after the adapting dose. The cells pre-exposed to low doses of gamma-rays showed cross-resistance to challenge doses of gamma-rays themselves and also of mitomycin C (MMC) and near ultraviolet light (UV-B, 313 nm), but not to those of ethyl methanesulfonate (EMS) or cis-platinum (II) diammine dichloride (cisplatin) for SCE induction. These results suggest that the radio-adaptive response mechanistically couples to the repair network which copes with chromatin lesions induced by MMC and UV-B.     

G2 repair and chromosomal damage in lymphocytes from workers occupationally exposed to low-level ionizing radiation

The effect of the G2 repair of chromosomal damage in lymphocytes from workers exposed to low levels of X- or gamma-rays was evaluated. Samples of peripheral blood were collected from 15 radiation workers, 20 subjects working in radiodiagnostics, and 30 healthy control donors. Chromosomal aberrations (CA) were evaluated by scoring the presence of chromatid and isochromatid breaks, dicentric and ring chromosomes in lymphocytes with/without 5 mM caffeine plus 3 mM-aminobenzamide (3-AB) treatment during G2. Our results showed that the mean value of basal aberrations in lymphocytes from exposed workers was higher than in control cells (p < 0.001). The chromosomal damage in G2, detected with caffeine plus 3-AB treatment was higher than the basal damage (untreated conditions), both in control and exposed populations (p < 0.05). In the exposed workers group, the mean value of chromosomal abnormalities in G2 was higher than in the control (p < 0.0001). No correlation was found between the frequency of chromosome type of aberrations (basal or in G2), and the absorbed dose. Nevertheless, significant correlation coefficients (p < 0.05) between absorbed dose and basal aberrations yield (r = 0.430) or in G2 (r = 0.448) were detected when chromatid breaks were included in the total aberrations yield. Under this latter condition no significant effect of age, years of employment or smoking habit on the chromosomal aberrations yield was detected. However, analysis of the relationship between basal aberrations yield and the efficiency of G2 repair mechanisms, defined as the percentage of chromosomal lesions repaired in G2, showed a significant correlation coefficient (r = -0.802; p < 0.001). These results suggest that in addition to the absorbed dose, the individual G2 repair efficiency may be another important factor affecting the chromosomal aberrations yield detected in workers exposed to low-level ionizing radiation.     

Radiation-induced chromosomal aberrations in the bone marrow of mice exposed to various doses of gamma radiation

The occurrence of chromosomal aberrations was studied at 1–14 days post-exposure in female BALB/c mice exposed to various doses of gamma radiation. The frequency of abnormal cells, chromatid and chromosome breaks, dicentrics, centric rings, acentric fragments and total aberrations increased with exposure dose, and it was highest at 7 Gy. A peak was recorded on day 1 post-exposure with a gradual decline thereafter. The chromosomal aberration yield reached a nadir on day 14 post-irradiation, without restoration to the control level. The best fit for the present data was by a linear-quadratic relationship between dose of radiation and the frequency of chromosomal aberrations.     

Mutations and chromosomal aberrations in hMTH1-transfected and non-transfected TK6 cells after exposure to low dose rates of gamma radiation

The aim of the present study was to analyse the dose rate effect of gamma radiation at the level of mutations, chromosomal aberrations, and cell growth in TK6 cells with normal as well as reduced levels of hMTH1 protein. TK6 cells were exposed to gamma radiation at dose rates ranging from 1.4 to 30.0 mGy/h (chronic exposure) as well as 24 Gy/h (acute exposure). Cell growth, frequency of thymidine kinase mutants, and of chromosomal aberrations in painted chromosomes 2, 8, and 14 were analysed. A decline in cell growth and an increase in unstable-type chromosomal aberrations with increasing dose rate were observed in both cell lines. A dose rate effect was not seen on mutations or stable-type chromosomal aberrations in any of the two cell lines. Reduction in the hMTH1 protein does not influence the sensitivity of TK6 cells to gamma radiation. This result fits well with data of others generated with the same cell line.     

Sensitivity of rat testes to inhibitors of nucleic acid synthesis. III. The inheritance of mitomycin C-induced structural rearrangements of chromosomes.

The inheritance of mitomycin C-induced aberrations was studied. Examination of spermatogonia showed statistically significant differences between the progeny of the untreated and treated rats in terms of total aberrant cells and chromosomal structural rearrangements. With the exception of gaps, breaks and fragments, which showed no significant differences between the progeny of the two treatment groups, the rest of the aberrations scored revealed significant increases in their frequencies with an increase in daily doses of mitomycin C (MC). The profile of the aberrations demonstrated a high incidence of X and Y chromosome dissociations, multiple autosomal associations, hypodiploidy, and translocation. Translocations consisted of autosome-autoome, autosome-X chromosome, autosome-X-Y chromosome, and autosome-Y chromosome. It is suggested that the reduced number of offspring per female in the F1 was the consequence of inherited MC-induced chromosomal errors.     

Search for clastogenic factors in the plasma of locally irradiated individuals

In studies reported in the 1960s and in several investigations since, plasma from irradiated individuals was shown to induce chromosomal aberrations when transferred into normal blood cultures. In the present study, the aim was to investigate the occurrence of these clastogenic factors (CF) using markers representing DNA damage produced in reporter lymphocytes that are treated with plasma from locally exposed individuals. Blood plasma was obtained from clinical patients with benign conditions before and after they had received radiation to small treatment volumes. Three patient groups were studied: (I) marginal resected basal cell carcinoma, (II) painful osteoarthritis of the knee, and (III) painful tendinitis of the elbow or the heel. Patients in each treatment group obtained the same fractionated treatment regimen, ranging from a total dose of 40 Gy (8 × 5 Gy, 2 factions/week) to a very small volume (1-3.5 cm3) in group I to a total dose of 6 Gy (6 × 1 Gy, 2 fractions/week) for groups II and III (treatment volumes 800-1150 cm3 and 80-160 cm3, respectively). The presence of CF in the plasma was investigated through cytogenetic (chromosomal aberrations, micronuclei) assays and kinetics of early DNA damage (γ-H2AX foci) in reporter cells. With the experimental settings applied, local radiation exposure had no apparent effect on the induction of CF in patient plasma; no deviations in chromosomal aberrations or micronucleus or focus induction were observed in reporter cells treated with postexposure plasma with respect to pre-exposure samples when the mean values of the groups were compared. However, there was a large interindividual variation in the plasma-induced DNA-damaging effects. Steroid treatment of patients was demonstrated to be the most influential factor affecting the occurrence of plasma factors; plasma from patients treated with steroids led to significant reductions of γ-H2AX foci and reduced numbers of chromatid aberrations in reporter cells. In addition to the locally exposed patients, newly obtained plasma samples from three radiological accident victims exposed in 1994 were examined. In contrast to the patient data, a significant increase in chromosomal aberrations was induced with plasma from two accident victims.     

Effect of chronic exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin on sister chromatid exchange levels in peripheral lymphocytes of the Rhesus monkey

Frequencies of sister chromatid exchanges and chromosomal aberrations were examined in peripheral lymphocytes of Rhesus monkeys which had been fed a diet containing 25 parts per trillion 2,3,7,8-tetrachlorodibenzop-dioxin for a period of 4 years. When compared to non-exposed control animals, no significant differences were noted for either of these cytogenetic endpoints. In addition; there was not a significant difference in sister chromatid exchange response to a challenge dose of mitomycin C in cells from 2,3,7,8-tetrachlorodibenzop-dioxin exposed animals compared to controls. Our results confirm the lack of genotoxic effects associated with 2,3,7,8-tetrachlorodibenzop-dioxin exposure.Abbreviations MMC mitomycin C - PHAA phytohemagglutinin-p - PPT parts per trillion - SCE sister chromatid exchange - TCDD 2,3,7,8-tetrachlorodibenzo-p-dioxin     

Biological effects of ion beams in Nicotiana tabacum L.

The biological effects of ion beams on Nicotiana tabacum L., particularly the induction of chromosome aberrations, were investigated. Dry seeds were exposed to ¹²C⁵⁺, ⁴He²⁺ and ¹H⁺ beams with linear energy transfer (LET) ranging from 1 to 111 keV/μm and irradiated with gamma-rays. Ion beams were more effective in reducing germination and survival of the seeds than gamma-rays. The LD₅₀ for ¹²C⁵⁺ beams, ⁴He²⁺ beams and gamma-rays were 35, 60 and 500 Gy, respectively. The frequencies of mitotic cells with chromosome aberrations, such as chromosome bridges, acentric fragments and lagging chromosomes in the root tip cells of the exposed seeds, increased linearly with increasing doses. Relative biological effectiveness (RBE) values, based on the doses that induced a survival inhibition of 50% and a 10% frequency of aberrant cells, were 14.3–17.5 for the ¹²C⁵⁺ beams, 7.0–8.3 for the ⁴He²⁺ beams and 7.8 for the ¹H⁺ beams. Furthermore, the relative ratios of the chromosome aberration types were significantly different between the ion beam and the gamma-ray regimes: chromosome fragments were more frequent in the former, and chromosome bridges in the latter. Based on these results, we concluded that the repair process of initial lesions induced by ion beams may be different from that induced by low- LET radiation. Received: 29 October 1998 / Accepted in revised form: 25 March 1999     

Mutagenic effect of the cytostatic drug thaliblastine on rat bone marrow cells when administered alone and in combination with radiation

The cytogenetic analysis was performed in the bone marrow cells of Wistar rats treated with a therapeutic dose of thaliblastine (250 mg/kg) and exposed to gamma-rays (2 Gy). Thaliblastine alone induced chromosome aberrations and polyploid cells. The latter were the result of the stathmokinetic effect of the drug. In contrast to gamma-radiation of 2 Gy thaliblastine elicited a minor mutagenic effect. The cytogenetic effect of the combined treatment is greater than the sum of the two agents delivered separately, the maximum effect of radiation and thaliblastine being exhibited on the 8th and the 12th hour, respectively. The difference between the sum of aberrations after separate treatments and the yield of aberrations after the combined treatment is due to chromatid fragments.     

AT cells are not radiosensitive for simple chromosomal exchanges at low dose

Cells deficient in ATM (product of the gene that is mutated in ataxia telangiectasia patients) or NBS (product of the gene mutated in the Nijmegen breakage syndrome) show increased yields of both simple and complex chromosomal aberrations after high doses (>0.5Gy) of ionizing radiation (X-rays or γ-rays), however less is known on how these cells respond at low dose. Previously we had shown that the increased chromosome aberrations in ATM and NBS defective lines was due to a significantly larger quadratic dose-response term compared to normal fibroblasts for both simple and complex exchanges. The linear dose-response term for simple exchanges was significantly higher in NBS cells compared to wild type cells, but not for AT cells. However, AT cells have a high background level of exchanges compared to wild type or NBS cells that confounds the understanding of low dose responses. To understand the sensitivity differences for high to low doses, chromosomal aberration analysis was first performed at low dose-rates (0.5Gy/d), and results provided further evidence for the lack of sensitivity for exchanges in AT cells below doses of 1Gy. Normal lung fibroblast cells treated with KU-55933, a specific ATM kinase inhibitor, showed increased numbers of exchanges at a dose of 1Gy and higher, but were similar to wild type cells at 0.5Gy or below. These results were confirmed using siRNA knockdown of ATM. The present study provides evidence that the increased radiation sensitivity of AT cells for chromosomal exchanges found at high dose does not occur at low dose.     

Kinetics of chromosomal aberrations and first mitosis division in human lymphocytes exposed to mitomycin C

In order to understand the relationship between the chromosomal damage detectable at the first mitosis after mutagen treatment and the induced mitotic delay we studied the time pattern of both mitotic indices and chromosomal aberration frequencies in human lymphocytes treated in G1 with mitomycin C (2.5 microM) and cultured in vitro in the presence of 5-bromo-2'-deoxyuridine. Mitotic delay was observed in treated cells cultured for 81 h. At this point an increase in the frequency of chromosomal aberrations is evident and a higher proportion of abnormal cells enters mitosis, the long delay being due to the extensiveness of DNA damage. The importance of cell cycle progression for the detection of the maximal amount of induced chromosomal damage is discussed.