Yield Of Dicentrics Research Articles

Kinetics of the Formation of Chromosome Aberrations in X-Irradiated Human Lymphocytes: Analysis by Premature Chromosome Condensation with Delayed Fusion

Human lymphocytes irradiated with graded doses of up to 5 Gy of 150 kV X rays were fused with mitotic CHO cells after delay times ranging from 0 to 14 h after irradiation. The yields of dicentrics seen under PCC conditions, using C-banding for centromere detection, and of excess acentric fragments observed in the PCC experiment were determined by image analysis. At 4 Gy the time course of the yield of dicentrics shows an early plateau for delay times up to 2 h, then an S-shaped rise and a final plateau which is reached after a delay time of about 8 to 10 h. Whereas the dose-yield curve measured at zero delay time is strictly linear, the shape of the curve obtained for 8 h delay time is linear-quadratic. The linear yield component, alpha D, is formed entirely in the fast process manifested in the early plateau, while component beta D2 is developed slowly in the subsequent hours. Analysis of the kinetics of the rise of the S-shaped curve for yield as a function of time leads to the postulate of an "intermediate product" of pairwise DNA lesion interaction, still fragile when subjected to the stress of PCC, but gradually processed into a stable dicentric chromosome. It is concluded that the observed difference in the kinetics of the alpha and beta components explains a number of earlier results, especially the disappearance of the beta component at high LET, and opens possibilities for chemical and physical modification of the beta component during the extended formation process after irradiation observed here.

Comparison of γ-ray induced dicentric yields in human lymphocytes measured by conventional analysis and FISH

In an earlier work stable aberrations and dicentrics were determined by fluorescence in situ hybridization (FISH) after various doses of 137Cs γ-rays. No corresponding calibration curve for dicentrics is available for determinations in terms of the conventional analysis as performed in our laboratory, In view of the potential for the application of chromosome painting to human biological dosimetry, it is desirable to determine such a calibration curve and this and the comparison of the resulting data to those obtained in terms of the FISH method is the objective of the present communication. In the study it is found that the linear-quadratic dose response curves for dicentrics, that are determined by the two methods, are significantly different, although the different target sizes are accounted for. A similar problem was found earlier for X-rays. It does not appear that the difference is due to technical difficulties in the FISH method, that has been improved by employing in addition to the whole chromosome DNA probes, a pan-centrometric DNA probe

Chromosome Aberrations Induced in Human Lymphocytes after Partial-Body Irradiation

Chromosomal aberrations in peripheral blood lymphocytes obtained from two patients before and after they received one fraction of partial-body irradiation for palliative treatment were analyzed. Blood samples were taken 30 min and 24 h after radiation treatment. The yield of dicentrics obtained from case A 30 min after a partial-body (about 21%) treatment with 8 Gy was 0.066/cell, while the yield obtained 24 h after radiation treatment was 0.071/cell. The fraction of irradiated lymphocytes that reached metaphase at 52 h was 0.08 as evaluated by mixing cultures of in vitro irradiated and unirradiated blood. The yield of dicentrics for blood from case B 30 min after 6 Gy partial-body (about 24%) irradiation was 0.655/cell, while the yield 24 h after irradiation was 0.605/cell. The fraction of irradiated cells was 0.29. Estimation of doses and irradiated fractions for the two cases using the method proposed by Dolphin and the Qdr method is discussed. Although there was no significant difference between the mean yields of dicentrics per cell obtained 30 min and 24 h after radiation treatment, the data obtained at 24 h seemed more useful for the purpose of dose estimation. When a higher dose (8 Gy) was delivered to a smaller percentage of the body, underestimation of the dose was encountered.

Human chromosome-specific changes in a human-hamster hybrid cell line (A L) assessed by fluorescent in situ hybridization (FISH)

Purpose : To quantitatively assess all gamma-ray induced chromosomal changes confined to one human chromosome using fluorescence microscopy and in situ hybridization with a fluorescently labeled human chromosome specific nucleic acid probe. Methods and Materials : Synchronized human-hamser hybrid cells containing human chromosome 11 wer obtained by a modified mitotic shake-off procedure. G1 phase cells (> 95%)_ were irradiated with 137Cs gamma rays (0, 0.5, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0 and 10.0 Gy) at a dose rate of 1.1 Gy/min and mitotic cells colleted 16–20 h later; chromosomal spreads were prepared, denatured,and hybridized with a fluorescein-tagged nucleic acid probe against total human DNA. Chromosomes were examined by fluorescence microscopy and all categories of change involving the human chromosome 11 as target, recorded. Results : Overall, of the 3104 human-hamster hybrid cells examined, 82.1% were euploid, of which 88.6% contained one copy of human chromosome 11, 6.2% contained two copies, and 5.2% contained 0 copies. This is compatiblewith mitotic nondisjunction in a small fraction of cells. Of the remaining 17.9% of cells, 85.2% were tetraploid cells with two copies of human chromosome 11. For all aberrations involving human chromosome 11 there was a linear relationship between yield and absorbed dose of 0.1 aberrations per chromosoem per Gy. The yield of dicentrics, translocations, and terminal deletions that involve one lesion on the human chromosoem was linear, while the yield of interstitial deletions that arise from two interacting lesions on the human chromosoem was curvilinear. The frequencies of dicentrics and translocations were about equal, while there was a high (40–60%) incidence of incomplete exchanges between human and hamster chromosomes. Conclusion : Fluorescent in situ hybridization (FISH) procedures allow for the efficient detection of a broad range of induced changes in target chromosomes. Symmetrical exchanges induced in G1 (translocations) were readily scored and found to equate with the complementary asymmetrical exchanges (dicentrics). That is, nonlethal stable changes, which might be concern ini carcinogenic processes, complement lethal, unstable changes. Interstitial deletions that may contribute to the loss of antioncogenes as well as to lethality are also readily detected with enhanced levels detected at higher doses. The high level of induced terminal deletions and of incomplete dicentrics and translocations indicate a partial failure of interaction between lesions induced in human and hamster DNA, and suggest that such interspecies interactions lack the fidelity of intraspecies DNA lesion interactions. This suggest caution in the use of such model systems as indicators of human cell responsiveness.

Fluorescence in situ hybridization detection of chromosomal aberrations in human lymphocytes: applicability to biological dosimetry.

Human lymphocytes in G0 have been irradiated with X-ray doses from 0 to 4.0 Gy. Metaphase chromosomes 2, 3 and 5 and all centromeres were painted using fluorescence in situ hybridization (FISH) probe libraries. Dicentrics, centric rings and acentrics in the whole genome as well as translocations involving the painted chromosomes were recorded. The translocations were subdivided as complete or incomplete. Interstitial insertions and inversions were also noted. The observations were also recorded according to the Protocol for Aberration Identification and Nomenclature Terminology (PAINT) system of scoring. Given that the painted chromosomes comprise 20.4% of the genome it was found that the yield of bicoloured dicentrics was consistent with the yield of dicentrics in the whole genome. The yield of radiation-induced translocations was not significantly higher than that of bicoloured dicentrics. Of the translocations, 60% were complete and it was concluded that the majority of dicentrics and translocations are complete exchanges. Chromosome 5 took part in exchanges marginally more commonly than its length suggests, but it is not known if this is a property of the chromosome or whether it is a donor-dependent observation. The PAINT system of recording rearrangements was examined and the suggested numerical interpretation of this nomenclature was considered to be unsuitable for use in the estimation of dose for cases of accidental overexposure.

Influence of ara A on the formation of dicentrics in irradiated quiescent CHO cells.

CHO K1-cells, arrested in G0/G1 phase were irradiated with graded doses of 150-kV X-rays, and the dicentric chromosome aberrations in the first metaphase after reincubation in full growth medium were evaluated. When 500 microM ara A was administered at least 1 h before irradiation, maximal enhancement of the dicentric yield was obtained under 'immediate plating' conditions as well as under 'delayed plating' conditions, and under both conditions only the linear component alpha D of the aberration yield was affected. Since ara A had no effect upon the number of induced dsbs, as measured with PFGE at doses up to 100 Gy, the action pathway of ara A is suggested to be the repair/misrepair process manifested in the linear component of the dicentric yield. By varying the time at which either ara A at 37 degrees C was administered before irradiation or ara A at 4 degrees C was added after irradiation, it was observed that the repair step sensitive to ara A was completed within about 5 min after irradiation. This is in agreement with other observations of ara A action upon a fast repair step. The conversion of DNA single-strand breaks or base damage sites into double-strand breaks as a consequence of polymerase beta inhibition by ara A would explain the present results.

The Effectiveness of High Energy Neon Ions in Producing Chromosomal Aberrations in Human Lymphocytes

Abstract This paper presents the results of the observation of chromosomal aberrations in human lymphocytes following irradiation by 189 MeV 20Ne ions. Dicentrics were observed at the first metaphase and chromosomal breaks at early interphase using the premature chromosome condensation technique (PCC). Comparison of these measurements with previous work with low LET radiation, produced RBEm values of 0.3 and 0.6 for dicentrics at metaphase and for breaks in early interphase, respectively. Recently published work on the repair of double strand breaks using the PCC technique shows that following X irradiation, the dicentric yield does not alter with repair time but the breaks decrease with repair time with a time constant in the region of 1 or 2 h. More recently published data in which cultures of mixed irradiated and unirradiated cells were scored for both dicentrics at metaphase and breaks at early interphase show that interphase death affects the dicentric yield at metaphase but not the break yield at early interphase. These observations place restrictions on the acceptability of models of radiation action.

Generalised Microdosimetric Calculations of Cell-to-Cell Variance

Abstract The cell-to-cell variance of an endpoint gives extra information, which is highly model dependent and thus potentially useful for distinguishing between possible mechanisms. Some years ago, Virsik and Harder calculated variances of dicentric chromosome aberrations using two assumptions: first, that dicentrics are produced independently by different radiation tracks, and second, that the dicentric yield due to a single track is Poisson distributed. It is shown that the formalism can be generalised so that the Poisson assumption is dropped. This affects predictions of neutron induced variances quite markedly. For lesions formed directly, such as immediate, irremediable chromosomal breaks, the formalism is general. For lesions formed by pairwise damage interaction, such as exchange-type chromosomal aberrations, the first assumption (single track action) limits the applicability of the results to high LET or low dose situations. In general, therefore, estimating variances requires the use of spatially inhomogeneous models.

Relative biological effectiveness and dose rate effect of tritiated water on chromosomes in human lymphocytes and bone marrow cells

Tritriated water (HTO) is a major toxic effluent from the nuclear power industry, that is released into the environment in large quantities. The low dose radiation effect and dose rate effect of HTO on human lymphocytes and bone marrow cells have not been well studied. The present study was therefore undertaken to investigate the HTO dose-response relationship for chromosomal aberrations in human lymphocytes and bone marrow cells at low in vitro radiation doses ranging from 0.1 to 1 Gy. Lymphocytes (G 0 stage) and bone marrow cells were incubated for 10–150 min with HTO at a dose rate of 2cGy/min (555 MBq/ml). The relative biological effectiveness (RBE) of HTO was calculated with respect to 60Co γ-rays for the induction of dicentric and centric ring chromosomes at low radiation doses. The RBE value for HTO β-rays relative to 60Co γ-rays was 2.7 for lymphocytes and 3.1 for chromatid aberrations in bone marrow cells. Lymphocytes were also chronically exposed to HTO for 6.7–80 h at dose rates of 0.5 cGy/min (138.5 MBq/ml) and 0.02 cGy/min (5.6 MBq/ml). There was a 71.5% decrease in the yield of dicentrics and centric rings at the dose rate of 0.02 cGy/min, indicating a clear dose rate effect of HTO. The RBE value for HTO relative to 137Cs γ-rays was 2.0 at a dose rate of 0.02 cGy/min, suggesting that low HTO dose rates produce no increase of the RBE values and that the values may be constant between 2 and 3 within these dose rates. These results should prove useful in assessment of the health risk for humans exposed to low levels of HTO.

Effects of colcemid concentration on chromosome aberration analysis in human lymphocytes.

As a part of technical improvements of chromosome aberration analysis on human peripheral lymphocytes for biological radiation dosimetry, we examined the optimal conditions for the use of colcemid in chromosome preparation in order to obtain enough number of cells at metaphase in the first cell division. When treated with colcemid at concentrations below 0.01 microgram/ml from the beginning of culture, cultures harvested at 48 hours had low mitotic indices. Colcemid treatment at 0.025 to 0.05 microgram/ml during 48 hours resulted in high mitotic indices (8 to 15%) and almost of the mitotic cells remaining in the 1st cell division, suggesting that this range of colcemid concentration was appropriate for continuous treatment with colcemid. We further examined the effect of colcemid concentration on the quantitative consistency of the yields of radiation-induced chromosome aberration. Repeated experiments showed that the yield of dicentrics and centric rings in the culture having colcemid at 0.025 microgram/ml concentration were larger than that at 0.05 microgram/ml. These data indicate the importance of assuring the accuracy of colcemid concentration in the lymphocyte culture for cytogenetic radiation dosimetry.

Effects of 252Cf neutrons, transmitted through an iron block on human lymphocyte chromosome.

Chromosome aberration of human peripheral blood lymphocytes exposed to californium-252 (252Cf) neutrons transmitted through a 15 cm thick iron block was analysed. The spectrum of the filtered neutrons ranged from 0.1 to 2 MeV with a peak at 0.7 MeV, simulating the Hiroshima atomic bomb neutron spectrum as shown in the Dosimetry System 1986 (DS86). Chromosome aberration frequencies after exposure to filtered and unfiltered 252Cf radiation were compared. Acentric ring chromosomes were significantly increased (p < 0.05) in the filtered condition. However, yields of dicentrics and centric rings induced by the filtered neutrons were not statistically different from those induced by the unfiltered neutrons (p > 0.1). The relative biological effectiveness (RBE) of the neutrons with respect to the formation of dicentrics and centric rings was 10.9 and 12.3 in the filtered and unfiltered conditions respectively, but the difference was not statistically significant. These results provide useful information for the re-evaluation of the biological effect of the Hiroshima atomic bomb radiations.

Modelling of DNA breaks and the formation of chromosome aberrations.

We present results of some calculations that convert double-strand breaks to dicentrics by means of a repair-misrepair model. The method uses Monte Carlo techniques to determine the positions of the double-strand breaks and to determine the dicentric yields for comparison with those obtained experimentally in human lymphocytes following irradiation in G0. The model is able to describe the linear-quadratic curves of dicentric yield following low LET irradiation, but underpredicts by a factor of about 1.5-2.0 the high LET neutron curves. The effects of including corrections for interphase death are demonstrated. The reasons for the discrepancy at high LET are discussed and suggestions for an improvement to the fit are made.

DNA polymerase δ mediates increase in exchange production by X-radiation in human lymphocytes moving from G0 to G1

Earlier work of several laboratories established that the yields of radiation-induced ring and dicentric chromosomes are greater when human peripheral blood lymphocytes are irradiated in GH1 some hours after phytohemagglutinin stimulation than if they are irradiated in G0 before stimulation. Post-treatment of lymphocytes irradiated in G0 with the DNA polymerase inhibitor aphidicolin, which is effective against both pol α and pol δ, produces a similar increase in ring and dicentric yield. We found that aphidicolin post-treatment was much less effective in increasing ring and dicentric yield increases in cells irradiated in G1 four to five hours after stimulation. Because we had earlier found specific inhibitors of DNA pol α ineffective in producing increased yields in either G0 or G1 lymphocytes, we conclude that much of the G0 to G1 increase in yields is mediated by pol δ.

Radiation-induced chromosomal breakage and rejoining in interphase-metaphase chromosomes of human lymphocytes

The premature chromosome condensation (PCC) technique and conventional chromosome analysis were applied to examine the kinetics of radiatio-induced primary chromosome breaks, their rejoining and formation of dicentrics in human peripheral blood lymphocytes (PBLs). Numbers of chromosomal elements and dicentrics per cell were analyzed for each dose. Dose-dependent increases were observed for chromosome fragments (linear) and dicentrics (linear/quadratic). For an assessment of repair kinetics, numbers of breaks and dicentrics were estimated immediately and at several recovery periods after irradiation, using the PCC technique. It was found that chromosome fragments restitute with time, whereas the dicentrics are formed very quickly and their frequency remains the same, despite the decline in the number of chromosome breaks at later recovery times. Fractionation experiments were conducted to study the time-dependent interaction of primary breaks in the formation of dicentric chromosomes. PBLs were irradiated with 3 Gy X-rays split into 2 equal fractions separated by different intervals up to 5 h. No marked difference was observed in the yield of dicentrics following the different fractionation protocols, except that themean levels of dicentrics declined when the fraction interval was 4 h or more. It appears that most of the dicentrics are formed by misrepair of strand breaks, produced directly by radiation and not resulting from rejoining of existing breaks during the slow repair process which follows. We also studied the role of the chromatin configuration at the time of irradiation on the yield of chromosome fragments and dicentrics. Highly condensed chromatin due to pretreatment with 0.3 M NaCl was found to reduce the frequency of radiation-induced chromosomal aberrations.

Reaction Kinetics for the Development of Radiation-induced Chromosome Aberrations

The formation of chromosome aberrations from DNA double-strand breaks (dsb) following ionizing irradiation of cells is analysed using a stochastic, continuous-time Markov chain formalism. A restitution/complete exchange model is proposed which incorporates kinetic competition between dsb restitution and chromosome exchange; it applies primarily to those dsb whose broken ends are held in close proximity by proteins. Some additional pathways for damage evolution are also considered. The calculations are compared in detail to the experiments on dicentric yield and variance in human lymphocytes following acute low-LET irradiation summarized by Lloyd and Edwards (1983) and Lloyd et al. (1987). It was found that dicentric formation by pairwise dsb interactions can lead to a dicentric yield/dose curve with a significant linear component even if one-track action is neglected. In other words a dicentric formation rate quadratic in the dsb number does not automatically imply a dicentric yield quadratic in the dose. However, the data indicate that at least in some experiments there could have been a significant one-track contribution to the dicentric yield in addition to the intertrack contributions analysed in the present paper. From the data the order of magnitude of the dsb interaction rate constant is estimated to be at most approximately 3 x 10(-3) exchanges per dsb pair per hour. It was found that for the reaction pathways considered, a initial Poisson distribution of dsb leads to an underdispersed distribution of dicentrics; the amount of underdispersion is strongly model-dependent and the variance data are consistent with the restitution/complete exchange model. Finally, a general mathematical theorem on Markov models is presented. It implies that assays performed after repair and exchange are completed cannot determine absolute repair or exchange rates, only their ratio (which may depend on lesion number).

Chromosome-type exchange aberrations are induced by inhibiting repair of IVC-induced DNA lesions in quiescent human lymphocytes

Human lymphocytes in the quiescent stage were UVC-irradiated and then incubated for 90 min in the presence of the DNA-repair inhibitor ara-C. The cells were then cultured and analyzed for chromosome aberrations. A single treatment with UVC or ara-C gives rise to a very low yield of dicentrics, whereas the combined treatment can induce a high frequency of these chromosome-type aberrations. The yield in the combined treatment is approximately proportionaal to the square of the UVC fluence in the range 1–3 J/m 2. In addition, the experiments demonstrate that synergistic effects arise when cells are treated with UVC + ara-C and then exposed to X-rays. The results can be explained on the assumption that the UVC + ara-C treatment induces DNA double-strand breaks which, to the first approximation, are randomly distributed over the chromosomes. These breaks are able to interact with each other as well as with X-ray-induced DNA double-strand breaks to form a chromosome-type exchange aberration.

The Induction of Chromosome Aberrations in Human Lymphocytes by 24 keV Neutrons

Abstract This paper describes an experiment to measure the yield of chromosome aberrations in human lymphocytes produced by a beam of predominantly 24 keV neutrons. The yield of dicentrics is linear with dose with a slope coefficient a of 0.821 ±0.031 dicentrics. cell-1. Gy-1. This coefficient is very close to that observed for fission neutrons of mean energy of 0.9 MeV. The main interaction of 24 keV neutrons with tissue is the production of protons of energy 24 keV and less which have a maximum range of 0.5 µm. Thus the experiment investigates the biological effectiveness of the track ends of higher energy protons. Previously inferred predictions of the relationships between the coefficient a and LET and between a and lineal energy, y, determined in 1 µm spheres, showed that analysis based on LET is much superior to that based on y. The findings are consistent with results using low energy X rays to investigate the biological effects of short range low energy electron tracks. Both experiments indicate that energy deposited in volumes of dimension much smaller that 1 µm is relevant for explaining biological effects.

The Induction of Chromosome Aberrations in Human Lymphocytes by 24 keV Neutrons

This paper describes an experiment to measure the yield of chromosome aberrations in human lymphocytes produced by a beam of predominantly 24 keV neutrons. The yield of dicentrics is linear with dose with a slope coefficient a of 0.821 ±0.031 dicentrics. cell-1. Gy-1. This coefficient is very close to that observed for fission neutrons of mean energy of 0.9 MeV. The main interaction of 24 keV neutrons with tissue is the production of protons of energy 24 keV and less which have a maximum range of 0.5 µm. Thus the experiment investigates the biological effectiveness of the track ends of higher energy protons. Previously inferred predictions of the relationships between the coefficient a and LET and between a and lineal energy, y, determined in 1 µm spheres, showed that analysis based on LET is much superior to that based on y. The findings are consistent with results using low energy X rays to investigate the biological effects of short range low energy electron tracks. Both experiments indicate that energy deposited in volumes of dimension much smaller that 1 µm is relevant for explaining biological effects.

Correlation between the anti-oxidant enzyme activities of blood fractions and the yield of bleomycin-induced chromosome damage

The concentration of the anti-oxidant enzymes catalase (CAT), peroxidases (POD) and superoxide dismutates (SOD) in different blood fractions, and the chromosomal sensitivity of lymphocytes to bleomycin-induced free radicals (expressed as frequency of dicentrics per bleomycin dose) were analyzed in 10 normal human donors. Our results demonstrate that the physiological concentration of the enzymes as well as the chromosomal sensitivity exhibited a wide interindividual variability. An inverse correlation between chromosomal sensitivity to bleomycin and SOD concentration in whole blood, plasma and red cells was found. On the other hand, no correlation between the yield of bleomycin-induced dicentrics and the concentration of CAT or POD was detected in any of the blood fractions analyzed. These findings suggest that the concentration of SOD may play an important role in the cellular susceptibility to DNA damage by free radicals.

Chromosomal aberrations and DNA repair ability of in vitro irradiated white blood cells of monkeys previously exposed to total body irradiation

Six monkeys ( Macaca fascicularis) were total-body-irradiated with 60Co (fractionated irradiation of 8 or 10 Gy). Blood samples were collected at different times post total-body irradiation, then in vitro irradiated in order to test whether a prior in vivo irradiation could affect the radiosensitivity of their leukocytes. We suggested in a preliminary report that the enhanced chromosomal radiosensitivity of in vivo irradiated monkeys could be correlated with a DNA repair deficiency (Guedeney et al., 1986). chromosomal aberrations, the rate of initial strand breaks and their rejoining estimated using a fluorescent assay for DNA unwinding were chosen as the endpoints in this more extensive study. We observed that the yield of dicentrics induced by a subsequent in vitro irradiation was lower than that scored in unirradiated monkeys in few cases (6/22) whereas the number of acentrics was found to be modified in 16 of the 22 samples. An altered DNA repair ability was observed in most but not all blood samples tested. Thus, in view of such intra-individual variability, the results of this more extensive study lead us to conclude that a previous total-body irradiation does not alter the γ-induced chromosome aberrations and DNA repair ability in a reproducible manner.