Biological effectiveness of accelerated particles for the induction of chromosome damage measured in metaphase and interphase human lymphocytes

Chromosome aberrations were investigated in human lymphocytes after in vitro exposure to 1H-, 3He-, 12C-, 40Ar-, 28Si-, 56Fe-, or 197Au-ion beams, with LET ranging from approximately 0.4-1393 keV/microm in the dose range of 0.075-3 Gy. Dose-response curves for chromosome exchanges, measured at the first mitosis postirradiation using fluorescence in situ hybridization (FISH) with whole-chromosome probes, were fitted with linear or linear-quadratic functions. The relative biological effectiveness (RBE) was estimated from the initial slope of the dose-response curve for chromosomal damage with respect to low- or high-dose-rate gamma rays. Estimates of RBEmax values for mitotic spreads, which ranged from near 0.7 to 11.1 for total exchanges, increased with LET, reaching a maximum at about 150 keV/microm, and decreased with further increase in LET. RBEs for complex aberrations are undefined due to the lack of an initial slope for gamma rays. Additionally, the effect of mitotic delay on RBE values was investigated by measuring chromosome aberrations in interphase after chemically induced premature chromosome condensation (PCC), and values were up to threefold higher than for metaphase analysis.     

Cellular and molecular effects for mutation induction in normal human cells irradiated with accelerated neon ions

We investigated the linear energy transfer (LET) dependence of mutation induction on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in normal human fibroblast-like cells irradiated with accelerated neon-ion beams. The cells were irradiated with neon-ion beams at various LETs ranging from 63 to 335 keV/microm. Neon-ion beams were accelerated by the Riken Ring Cyclotron at the Institute of Physical and Chemical Research in Japan. Mutation induction at the HPRT locus was detected to measure 6-thioguanine-resistant clones. The mutation spectrum of the deletion pattern of exons of mutants was analyzed using the multiplex polymerase chain reaction (PCR). The dose-response curves increased steeply up to 0.5 Gy and leveled off or decreased between 0.5 and 1.0 Gy, compared to the response to (137)Cs gamma-rays. The mutation frequency increased up to 105 keV/microm and then there was a downward trend with increasing LET values. The deletion pattern of exons was non-specific. About 75-100% of the mutants produced using LETs ranging from 63 to 335 keV/mum showed all or partial deletions of exons, while among gamma-ray-induced mutants 30% showed no deletions, 30% partial deletions and 40% complete deletions. These results suggested that the dose-response curves of neon-ion-induced mutations were dependent upon LET values, but the deletion pattern of DNA was not.     

Effect of linear energy transfer (LET) on the complexity of alpha-particle-induced chromosome aberrations in human CD34+ cells

The aim of this study was to assess the relative influence of the linear energy transfer (LET) of alpha particles on the complexity of chromosome aberrations in the absence of significant other differences in track structure. To do this, we irradiated human hemopoietic stem cells (CD34+) with alpha particles of various incident LETs (110-152 keV/microm, with mean LETs through the cell of 119-182 keV/microm) at an equi-fluence of approximately one particle/cell and assayed for chromosome aberrations by mFISH. Based on a single harvest time to collect early-division mitotic cells, complex aberrations were observed at comparable frequencies irrespective of incident LET; however, when expressed as a proportion of the total exchanges detected, their occurrence was seen to increase with increasing LET. Cycle analysis to predict theoretical DNA double-strand break rejoining cycles was also carried out on all complex chromosome aberrations detected. By doing this we found that the majority of complex aberrations are formed in single non-reducible cycles that involve just two or three different chromosomes and three or four different breaks. Each non-reducible cycle is suggested to represent "an area" of finite size within the nucleus where double-strand break repair occurs. We suggest that the local density of damage induced and the proximity of independent repair areas within the interphase nucleus determine the complexity of aberrations resolved in metaphase. Overall, the most likely outcome of a single nuclear traversal of a single alpha particle in CD34+ cells is a single chromosome aberration per damaged cell. As the incident LET of the alpha particle increases, the likelihood of this aberration being classed as complex is greater.     

The cytogenetic effects of low doses of accelerated charged particles in human blood lymphocytes in vitro

The aim of the investigation was the study of cytogenetic effects in human blood lymphocytes of low doses of ionizing radiation in vitro. The analysis of unstable chromosome aberrations in human lymphocytes after irradiation by the accelerated ions 12C with the energy 500 MeV/nucleon and LET 10.7 keV/microm was carried out. Blood samples were irradiated on Nuclotron of the High Energy Laboratory of the Joint Institute for Nuclear Research. The doses of irradiation were in the range from 0.05 up to 1.0 Gy. Was shown that the frequency of unstable chromosome aberrations depends from the dose of ionizing radiation and can be described by linear function. At the doses 0.25-0.50 Gy the dose-independent curve was obtained for dicentrics and centric rings. The frequencies of dicentrics and centric rings as markers of the radiation action were slightly different for different donors that could be explained by different radiosensitivity. Using the calibration curve obtained earlier for gamma-rays coefficients of relative biological efficiency of accelerated 12C with the energy 500 MeV/nucleon were defined: they varied from 1.0 at the doses (0.5-1.0 Gy) up to 3.2 at the lower doses (0.05-0.25 Gy).     

Induction of chromosome aberrations and micronuclei in human peripheral blood lymphocytes at low dose of radiation

The chromosome damage induced by the doses of y-irradiation 6)Co in peripheral blood lymphocytes was studied using different cytogenetic assays. Isolated lymphocytes were exposed to 0.01-1.0 Gy, stimulated by PHA, and analysed for chromosome aberrations at 48 h postirradiation by metaphase method, at 49 h--by the anaphase method, at 58 h by micronucleus assay with cytochalasin B and, additionally, micronuclei were counted at 48 h on the slides prepared for the metaphase analysis without cytochalasin B. Despite of the quantitative differences in the amount of chromosome damage revealed by different methods all of them demonstrated complex nonlinear dose dependence of the frequency of aberrant cells and aberrations. At the dose range from 0.01 Gy to 0.05-0.07 Gy the cells had the highest radiosensitivity mainly due to chromatid-type aberration induction. With dose increasing the frequency of the aberrant cells and aberrations decreased significantly (in some cases to the control level). At the doses up to 0.5-0.7 Gy the dose-effect curves have become linear with the decreased slope compare to initial one (by factor of 5 to 10 for different criteria) reflecting the higher radioresistance of cells. These data confirm the idea that the direct linear extrapolation of high dose effect to low dose range--the procedure routinelly used to estimate genetic risk of low dose irradiation--cannot be effective and may lead to underestimation of chromosome damage produced by low radiation doses. Preferences and disadvantages of used cytogenetic assays and possible mechanisms of low ionising radiation doses action were discussed.     

Relative biological effectiveness of high-energy iron ions for micronucleus formation at low doses

Dose-response curves for micronucleus (MN) formation were measured in Chinese hamster V79 and xrs6 (Ku80(-)) cells and in human mammary epithelial MCF10A cells in the dose range of 0.05-1 Gy. The Chinese hamster cells were exposed to 1 GeV/nucleon iron ions, 600 MeV/nucleon iron ions, and 300 MeV/nucleon iron ions (LETs of 151, 176 and 235 keV/microm, respectively) as well as with 320 kVp X rays as reference. Second-order polynomials were fitted to the induction curves, and the initial slopes (the alpha values) were used to calculate RBE. For the repair-proficient V79 cells, the RBE at these low doses increased with LET. The values obtained were 3.1 +/- 0.8 (LET = 151 keV/microm), 4.3 +/- 0.5 (LET = 176 keV/microm), and 5.7 +/- 0.6 (LET = 235 keV/microm), while the RBE was close to 1 for the repair-deficient xrs6 cells regardless of LET. For the MCF10A cells, the RBE was determined for 1 GeV/nucleon iron ions and was found to be 5.5 +/- 0.9, slightly higher than for V79 cells. To test the effect of shielding, the 1 GeV/nucleon iron-ion beam was intercepted by various thicknesses of high-density polyethylene plastic absorbers, which resulted in energy loss and fragmentation. It was found that the MN yield for V79 cells placed behind the absorbers decreased in proportion to the decrease in dose both before and after the iron-ion Bragg peak, indicating that RBE did not change significantly due to shielding except in the Bragg peak region. At the Bragg peak itself with an entrance dose of 0.5 Gy, where the LET is very high from stopping low-energy iron ions, the effectiveness for MN formation per unit dose was decreased compared to non-Bragg peak areas.     

Chromatid-type aberrations following irradiation in G0 lymphocytes with heavy ions

We describe a low level of chromatid-type aberrations which included the relatively rare isochromatid/chromatid triradial in peripheral blood lymphocytes that were irradiated, ostensibly in G0, with accelerated heavy (12)C ions. These were produced only at the energies of 69 MeV/n (34.6 keV/microm), almost absent at the energy of either 58.6 MeV/n (46.07 keV/ microm) or 19.3 MeV/n (97 keV/microm), nor were they found after low-LET X-rays. Mechanisms potentially responsible for their formation are discussed.     

Investigation of radiation-induced "bystaner effect" using model of adaptive response in mixed lymphocyte culture from humans of different gender

The novel method for the investigation of radiation-induced "bystander effect" has been tested on the model of mixed lymphocyte culture from humans of different gender. The "bystander effect" was estimated by the ability of nonirradiated female/male cells to develop an adaptive response in mixed culture with irradiated at the dose 0.05 Gy of X-rays G0 lymphocytes of opposite gender. The preliminary results indicate that both irradiated lymphocytes and non-irradiated but neighbouring with pre-exposed cells are less susceptible to the genetic damages manifested as chromosome aberrations induced in G1 lymphocytes by a subsequent high dose of X-ray (1.0 Gy). Direct adaptive response as well as indirect one were expressed more obvious in female lymphocytes.     

The genotoxicity of alpha particles in human embryonic skin fibroblasts

Cell inactivation and induced mutation frequencies at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus have been measured in cultured human fibroblasts (GM10) exposed to alpha particles from 238Pu (LET at the cell surface was 100 keV/microns) and 250 kVp X rays. The survival curves resulting from exposure to alpha particles are exponential. The mean lethal dose, D0, is approximately 1.3 Gy for X rays and 0.25 Gy for alpha particles. As a function of radiation dose, mutation induction at the HGPRT locus was linear for alpha particles whereas the X-ray-induced mutation data were better fitted by a quadratic function. When mutation frequencies were plotted against the log of survival, mutation frequency at a given survival level was greater in cells exposed to alpha particles than to X rays.     

A model for interphase chromosomes and evaluation of radiation-induced aberrations

We have developed a theoretical model for evaluating radiation-induced chromosomal exchanges by explicitly taking into account interphase (G(0)/G(1)) chromosome structure, nuclear organization of chromosomes, the production of double-strand breaks (DSBs), and the subsequent rejoinings in a faithful or unfaithful manner. Each of the 46 chromosomes for human lymphocytes (40 chromosomes for mouse lymphocytes) is modeled as a random polymer inside a spherical volume. The chromosome spheres are packed randomly inside a spherical nucleus with an allowed overlap controlled by a parameter Omega. The rejoining of DSBs is determined by a Monte Carlo procedure using a Gaussian proximity function with an interaction range parameter sigma. Values of Omega and sigma have been found which yield calculated results of interchromosomal aberration frequencies that agree with a wide range of experimental data. Our preferred solution is one with an interaction range of 0.5 microm coupled with a relatively small overlap parameter of 0.675 microm, which more or less confirms previous estimates. We have used our model with these parameter values and with resolution or detectability limits to calculate yields of translocations and dicentrics for human lymphocytes exposed to low-LET radiation that agree with experiments in the dose range 0.09 to 4 Gy. Five different experimental data sets have been compared with the theoretical results. Essentially all of the experimental data fall between theoretical curves corresponding to resolution limits of 1 Mbp and 20 Mbp, which may reflect the fact that different investigators use different limits for sensitivity or detectability. Translocation yields for mouse lymphocytes have also been calculated and are in good agreement with experimental data from 1 cGy to 10 cGy. There is also good agreement with recent data on complex aberrations. Our model is expected to be applicable to both low- and high-LET radiation, and we include a sample prediction of the yield of interchromosomal rejoining in the dose range 0.22 Gy to 2 Gy of 1000 MeV/nucleon iron particles. This dose range corresponds to average particle traversals per nucleus ranging from 1.0 to 9.12.     

Adaptive response to chromosome damage in cultured human lymphocytes primed with low doses of X-rays

Human lymphocytes exposed to 0.02 Gy of X-rays in the G1 but not the G0 phase became less susceptible to the induction of chromosome aberrations of the chromosome type by subsequent exposure to 3 Gy of X-rays. The induction of chromatid-type aberrations was not affected by the pretreatment with the priming dose. The expression of this adaptive-type response was transitory, being maximum at 5 h, and disappeared at 9 h after the initial low-dose exposure. Cell-cycle analysis excluded the possibility of a spurious consequence of differential cell-cycle progression.     

Transmissible and nontransmissible complex chromosome aberrations characterized by three-color and mFISH define a biomarker of exposure to high-LET alpha particles

Insertions have been proposed as potential stable biomarkers of chronic high-LET radiation exposure. To examine this in vitro, we irradiated human peripheral blood lymphocytes in G(0) with either 50 cGy (238)Pu alpha particles (LET 121.4 keV/microm) or 3 Gy 250 kV X rays and stimulated their long-term culture up to approximately 22 population doublings postirradiation. Mitotic cells were harvested at regular intervals throughout this culture period and were assayed for chromosome aberrations using the techniques of three-color and 24-color mFISH. We observed the stable persistence of transmissible-type complex rearrangements, all involving at least one insertion. This supports the hypothesis that insertions are relevant indicators of exposure to high-LET radiation. However, one practical caveat of insertions being effective biomarkers is that their frequency is low due to the complexity and cell lethality of the majority of alpha-particle-induced complexes. Therefore, we propose a "profile of damage" that relies on the presence of insertions, a low frequency of stable simple reciprocal translocations (2B), and, significantly, the complexity of the damage initially induced. We suggest that the complexity of first- and second-division alpha-particle-induced nontransmissible complex aberrations reflects the structure of the alpha-particle track and as a consequence adds radiation-quality specificity to the biomarker, increasing the signal:noise ratio of the characteristic 2B:insertion ratio.     

Biodosimetry estimate for high-LET irradiation

The purpose of this paper is to prepare for an easy and reliable biodosimeter protocol for radiation accidents involving high-linear energy transfer (LET) exposure. Human peripheral blood lymphocytes were irradiated using carbon ions (LET: 34.6 keV μm⁻¹), and the chromosome aberrations induced were analyzed using both a conventional colcemid block method and a calyculin A induced premature chromosome condensation (PCC) method. At a lower dose range (0–4 Gy), the measured dicentric (dics) and centric ring chromosomes (cRings) provided reasonable dose information. At higher doses (8 Gy), however, the frequency of dics and cRings was not suitable for dose estimation. Instead, we found that the number of Giemsa-stained drug-induced G2 prematurely condensed chromosomes (G2-PCC) can be used for dose estimation, since the total chromosome number (including fragments) was linearly correlated with radiation dose (r = 0.99). The ratio of the longest and the shortest chromosome length of the drug-induced G2-PCCs increased with radiation dose in a linear-quadratic manner (r = 0.96), which indicates that this ratio can also be used to estimate radiation doses. Obviously, it is easier to establish the dose response curve using the PCC technique than using the conventional metaphase chromosome method. It is assumed that combining the ratio of the longest and the shortest chromosome length with analysis of the total chromosome number might be a valuable tool for rapid and precise dose estimation for victims of radiation accidents.     

3-Aminobenzamide does not affect X-ray-induced cytogenetic damage in G0 human lymphocytes

The inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide (3AB) has been reported to have very different effects on X-ray-induced chromosome aberrations in G0 human lymphocytes. One group of investigators observed a 2-3-fold increase in the yield of rings, dicentrics and chromosome breaks after X-irradiation and 3AB treatment, whereas another group found that 3AB had no effect on X-ray-induced chromosome aberrations. To resolve this discrepancy, we repeated the experiments as described by both groups and found no effect of 3 mM or 5 mM 3AB on the frequency of chromosome aberrations induced by either 1 Gy or 2 Gy of X-rays. Furthermore, we found no effect of 3AB on X-ray-induced aberration yields in C-banded prematurely condensed chromosome preparations from unstimulated human lymphocytes. These results indicate that poly(ADP-ribose) polymerase is not involved in the repair of cytogenetic damage in G0 human lymphocytes.     

Chromosome aberrations produced in human lymphocytes by in vivo and in vitro irradiation

The production of chromosome aberrations in vivo has been studied in lymphocytes from a patient undergoing a wholebody treatment with gamma-radiation up to a cumulative dose of 1.4 Gy. These results were compared with the observations performed on whole blood samples irradiated in vitro with doses from 0.05 up to 2 Gy of gamma-rays. The frequency of chromosome aberrations, particularly the dicentrics, was found to be similar in vivo and in vitro. The yield of dicentrics could be best related to the dose by using a linear-quadratic model in both cases, the ratio of the coefficients a/b being of 0.56 and 0.69 Gy, respectively in vivo and in vitro. These observations confirm that in vitro dose response curves may be used to evaluate accurately an in vivo absorbed dose.     

Chromosome-type aberrations induced in chromosome 9 after treatment of human peripheral blood lymphocytes with mitomycin C at the G(0) phase.

To determine the fate of chromosome aberrations induced primarily by clastogenic chemicals, aberrations of chromosome 9 in cultured human peripheral blood lymphocytes were analyzed after exposure to mitomycin C (MMC) at G(0) phase. Chromosome 9 painting by fluorescence in situ hybridization revealed that the translocation of 9p or 9q to another chromosome and the centric fragment representing the entire length of 9p were characteristically generated from chromatid-type aberrations involving the centromeric region of chromosome 9. These changes were not observed at 48 h after culture initiation, but persistently appeared at later stages (72-120 h postinitiation). Induction of centric fragments of 9p and micronuclei without the alpha satellite DNA of chromosome 9 suggested that most of the breaks were induced near the alpha satellite DNA locus on 9q. Modified patterns of chromosome 9 aberrations were also observed, being related to the copy number of the short or long arm of the chromosome. Such unbalanced karyotypes could remain in the lymphocyte genome over further cell divisions for at least 120 h after culture initiation, indicating that these aberrant cells can survive and that they could pose a health risk.     

Radiation-induced "bystander effect" revealed by means of adaptive response in cocultured lymphocytes from humans of different genders

The "bystander effect" was investigated in mixed cultures of lymphocytes from humans of opposite genders. Development of the adaptive response (AR) in non-irradiated female/male cells was estimated after adaptive pretreatment of opposite gender lymphocytes, chromosome aberrations being evaluated. Experiments were performed using two schedules of adaptive (0.05 Gy) and challenging (1 Gy) irradiations: G0-G1 and G1-G1. The results obtained indicate the development of a mediated adaptive response ("bystander effect") in the lymphocytes neighboring pre-irradiated cells, as well as the influence of a time scheme of adapting and challenging irradiations on the amount of induced chromosome aberrations in mixed cultures and a possible dependence of the adaptive response intensity on the donor gender.     

Karyotypes of human lymphocytes exposed to high-energy iron ions

Chromosomal aberrations were analyzed using multicolor fluorescence in situ hybridization (mFISH) in human peripheral blood lymphocytes after in vitro exposure to gamma rays or accelerated (56)Fe ions (1 GeV/nucleon, 145 keV/microm) at Brookhaven National Laboratory (Upton, NY). Doses of 0.3 and 3 Gy were used for both radiation types. Chromosomes were prematurely condensed by a phosphatase inhibitor (calyculin A) to avoid the population selection bias observed at metaphase as a result of the severe cell cycle delays induced by heavy ions. A total of 1053 karyotypes (G(2) and M phases) were analyzed in irradiated lymphocytes. Results revealed different distribution patterns for chromosomal aberrations after low- and high-LET radiation exposures: Heavy ions induced a much higher fraction of cells with multiple aberrations, while the majority of the aberrant cells induced by low doses of gamma rays contained a single aberration. The high fraction of complex-type exchanges after heavy ions leads to an overestimation of simple-type asymmetrical interchanges (dicentrics) from analysis of Giemsa-stained samples. However, even after a dose of 3 Gy iron ions, about 30% of the cells presented no complex-type exchanges. The involvement of individual chromosomes in exchanges was similar for densely and sparsely ionizing radiation, and no statistically significant evidence of a nonrandom involvement of specific chromosomes was detected.     

Truly incomplete and complex exchanges in prematurely condensed chromosomes of human fibroblasts exposed in vitro to energetic heavy ions

Confluent human fibroblast cells (AG1522) were irradiated with gamma rays, 490 MeV/nucleon silicon ions, or iron ions at either 200 or 500 MeV/nucleon. The cells were allowed to repair at 37 degrees C for 24 h after exposure, and a chemically induced premature chromosome condensation (PCC) technique was used to condense chromosomes in the G2 phase of the cell cycle. Incomplete and complex exchanges were analyzed in the irradiated samples. To verify that chromosomal breaks were truly unrejoined, chromosome aberrations were analyzed using a combination of whole-chromosome specific probes and probes specific for the telomere region of the chromosome. Results showed that the frequency of unrejoined chromosome breaks was higher after irradiation with the heavy ions of high LET, and consequently the ratio of incomplete to complete exchanges increased steadily with LET up to 440 keV/microm, the highest LET included in the present study. For samples exposed to 200 MeV/nucleon iron ions, chromosome aberrations were analyzed using the multicolor FISH (mFISH) technique, which allows identification of both complex and truly incomplete exchanges. Results of the mFISH study showed that 0.7 and 3 Gy iron ions produced similar ratios of complex to simple exchanges and incomplete to complete exchanges; these ratios were higher than those obtained after exposure to 6 Gy gamma rays. After 0.7 Gy of iron ions, most complex aberrations were found to involve three or four chromosomes, which is a likely indication of the maximum number of chromosome domains traversed by a single iron-ion track.     

Dose-rate effect on the induction of HPRT mutants in human G0 lymphocytes exposed in vitro to gamma radiation

The influence of dose rate on expression time, cell survival and mutant frequency at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus was evaluated in human G(0) peripheral blood lymphocytes exposed in vitro to gamma rays at low (0.0014 Gy/min) and high (0.85 Gy/min) dose rates. A cloning assay performed on different days of postirradiation incubation indicated an 8-day maximum expression period for the induction of HPRT mutants at both high and low dose rates. Cell survival increased markedly with decreasing dose rate, yielding D(0) values of 3.04 Gy and 1.3 Gy at low and high dose rates, respectively. The D(0) of 3.04 Gy obtained at low dose rate could be attributed to the repair of sublethal DNA damage taking place during prolonged exposure to low-LET radiation. Regression analysis of the mutant frequency yielded slopes of 12.35 x 10(-6) and 3.66 x 10(-6) mutants per gray at high and low dose rate, respectively. A dose and dose-rate effectiveness factor of 3.4 indicated a marked dose-rate effect on the induced HPRT mutant frequency. The results indicate that information obtained from in vitro measurements of dose-rate effects in human G(0) lymphocytes may be a useful parameter for risk estimation in radiation protection.