Study of dose-rate effects of neutron radiation in a wild-type and a repair-deficient yeasts saccharomyces

We report here a comparative analysis of RBE for lethality of a single pulse (duration 65 micros) of fast neutron with ultra high dose rates (up to 6 x 10(6) Gy/s) and continuous neutron radiation (3.6 x 10(3) s) of the pulse reactor BARS-6. Three diploid strains, one haploid strain and three diploid repair-deficient strains (rad52-1/rad52-1; rad54/rad54; rad2/rad2) were used. The RBE values (D(0gamma)/1D(0n)) of a single pulse and continuous neutron irradiation were equal (1.7-1.8) with maximum RBE (4.1-3.1) in region of low doses (shoulder region). Haploid cells were found to be more (3 times) sensitive to both gamma-rays and neutrons than the wild type. There was no obvious decrease in the RBE of 1.9 in highly sensitive haploid cells as compared with highly resistant diploid cells. The repair-deficient strains (rad52-1/rad52-1; rad54/rad54) were more (up to 10 fold) sensitive to both neutrons and gamma-rays as compared with their parent line. The RBE values of 1.5-1.7 of neutrons for these mutants (independent by of the mode of irradiation) were found. The repair-deficient mutant rad2/rad2 had similar sensitivity as a wild type and a RBE value was 2.0. We have concluded that biological effectiveness of the neutrons of pulse reactor BARS-6 was independent of the dose-rate, differing up to 10(8) fold. The RBE didn't vary significantly with the capacity of cells to repair DNA damages.     

Radiolysis of liposomes, of solutions of ferrous sulphate and of albumin mixed by gamma-neutron radiation at different dose rates

Solutions of the ferrous sulfate, of the albumin and of the suspension of liposomes were irradiated by mixed gamma-neutron radiation (fission spectrum neutrons, contribution of gamma-component to the absorbed dose up to 20%) at the pulse reactor BARS-6 with single-pulse (duration 100 micros) or continuous radiation (duration 60 min). It was shown, that after the pulse irradiation the concentration of the malonic dialdehyde in liposomes was in 3-4 times higher than after the continuous radiation at equal absorbed doses (p < 0.05). On the contrary, the irradiation of the liposomes suspension as well as of the solutions of the ferrous sulfate and of the albumin in a mode of single-pulse or of continuous mode did not reveal the statistically significant differences in the production of Fe3+ ions and of peroxides of the albumin for two mode of the radiation action.     

High yields of isochromatid breaks and successive formation of chromosome exchanges may lead to reproductive cell death following high-LET irradiation

To clarify the relationship between cell death and chromosomal aberrations following exposure to heavy-charged ion particles beams, exponentially growing Human Salivary Gland Tumor cells (HSG cells) were irradiated with various kinds of high energy heavy ions; 13 keV/μm carbon ions as a low-LET charged particle radiation source, 120 keV/μm carbon ions and 440 keV/μm iron ions as high-LET charged particle radiation sources. X-rays (200 kVp) were used as a reference. Reproductive cell death was evaluated by clonogenic assays, and the chromatid aberrations in G2/M phase and their repairing kinetics were analyzed by the calyculin A induced premature chromosome condensation (PCC) method. High-LET heavy-ion beams introduced much more severe and un-repairable chromatid breaks and isochromatid breaks in HSG cells than low-LET irradiation. In addition, the continuous increase of exchange aberrations after irradiation occurred in the high-LET irradiated cells. The cell death, initial production of isochromatid breaks and subsequent formation of chromosome exchange seemed to be depend similarly on LET with a maximum RBE peak around 100–200 keV/μm of LET value. Conversely, un-rejoined isochromatid breaks or chromatid breaks/gaps seemed to be less effective in reproductive cell death. These results suggest that the continuous yield of chromosome exchange aberrations induced by high-LET ionizing particles is a possible reason for the high RBE for cell death following high-LET irradiation, alongside other chromosomal aberrations additively or synergistically.     

Effect of temperature on the development of chromosome aberrations in human blood lymphocytes irradiated in pulse and continuous modes at BARS-6 reactor

Summarized results of 5 repeated experiments of comparative study of radiation effects of the pulse reactor BARS-6 either in a single pulse or a continuos irradiation mode on human lymphocytes are presented. Higher efficiency (30-40%) of continuous irradiation (exposure duration 1 h) rather than pulse irradiation with ultra-high dose rates (1-2.5) x 10(6) Gy/min (pulse duration 65 micros) was confirmed. The efficiency ratio did not depend on the temperature, 20 degrees C or 0 degrees C, during the exposure. Cell repair system and chromatin conformation influence on the results obtained is discussed.     

Repair of DNA damage induced by accelerated heavy ions in mammalian cells proficient and deficient in the non-homologous end-joining pathway

Human and rodent cells proficient and deficient in non-homologous end joining (NHEJ) were irradiated with X rays, 70 keV/microm carbon ions, and 200 keV/microm iron ions, and the biological effects on these cells were compared. For wild-type CHO and normal human fibroblast (HFL III) cells, exposure to iron ions yielded the lowest cell survival, followed by carbon ions and then X rays. NHEJ-deficient xrs6 (a Ku80 mutant of CHO) and 180BR human fibroblast (DNA ligase IV mutant) cells showed similar cell survival for X and carbon-ion irradiation (RBE = approximately 1.0). This phenotype is likely to result from a defective NHEJ protein because xrs6-hamKu80 cells (xrs6 cells corrected with the wild-type KU80 gene) exhibited the wild-type response. At doses higher than 1 Gy, NHEJ-defective cells showed a lower level of survival with iron ions than with carbon ions or X rays, possibly due to inactivation of a radioresistant subpopulation. The G(1) premature chromosome condensation (PCC) assay with HFL III cells revealed LET-dependent impairment of repair of chromosome breaks. Additionally, iron-ion radiation induced non-repairable chromosome breaks not observed with carbon ions or X rays. PCC studies with 180BR cells indicated that the repair kinetics after exposure to carbon and iron ions behaved similarly for the first 6 h, but after 24 h the curve for carbon ions approached that for X rays, while the curve for iron ions remained high. These chromosome data reflect the existence of a slow NHEJ repair phase and severe biological damage induced by iron ions. The auto-phosphorylation of DNA-dependent protein kinase catalytic subunits (DNA-PKcs), an essential NHEJ step, was delayed significantly by high-LET carbon- and iron-ion radiation compared to X rays. This delay was further emphasized in NHEJ-defective 180BR cells. Our results indicate that high-LET radiation induces complex DNA damage that is not easily repaired or is not repaired by NHEJ even at low radiation doses such as 2 Gy.     

Pulsed-dose-rate and low-dose-rate brachytherapy: comparison of sparing effects in cells of a radiosensitive and a radioresistant cell line

Pulsed-dose-rate regimens are an attractive alternative to continuous low-dose-rate brachytherapy. However, apart from data obtained from modeling, only a few in vitro results are available for comparing the biological effectiveness of both modalities. Cells of two human cell lines with survival fractions of 80% (RT112) and 10% (HX142) after a single dose of 2 Gy and with different halftimes for split-dose recovery and low-dose recovery were used. The cells were irradiated with a continuous low dose rate (80 cGy per hour) or with pulsed dose rate. Two different pulsed dose rates were tested: 4.25 Gy/h and 63 Gy/h. The effects of dose per pulse and the length of the interval between the pulses were investigated while keeping the overall treatment time constant. Survival after low-dose-rate irradiation was indistinguishable from that after pulses of 4.25 Gy/h in cells of both cell lines. Survival decreased with increasing dose per pulse. When the dose rate during the pulses was increased, survival decreased even further. This effect was most pronounced for the radiosensitive HX142 cells. In clinical pulsed-dose-rate brachytherapy, iridium sources move stepwise through the implant and deliver pulses at a high dose rate locally. These high-dose-rate pulses produce greater biological effectiveness compared to continuous low dose rate; this should be taken into account.     

Ionizing radiation-induced instant pairing of heterochromatin of homologous chromosomes in human cells

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

The effectiveness of 20 mev protons at nanosecond pulse lengths in producing chromosome aberrations in human-hamster hybrid cells

Laser accelerated radiotherapy is a potential cancer treatment with proton and carbon-ion beams that is currently under development. Ultra-fast high-energy laser pulses will accelerate ion beams that deliver their dose to a patient in a "pulsed mode" that is expected to differ from conventional irradiation by increasing the dose delivery rate to a tissue voxel by approximately 8 orders of magnitude. In two independently performed experiments at the ion microprobe SNAKE of the 14 MV Munich tandem accelerator, A(L) cells were exposed either to protons with 1-ns pulse durations or to protons applied over 150 ms in continuous irradiation mode. A slightly but consistently lower aberration yield was observed for the pulsed compared to the continuous mode of proton irradiation. This difference was not statistically significant when each aberration type was analyzed separately (P values between 0.61 and 0.85 in experiment I and P values between 0.32 and 0.64 in experiment II). However, excluding the total aberrations, which were not analyzed as independent radiation-induced effects, the mean ratio of the yields of dicentrics, centric rings and excess acentrics scored together showed (with 95% CI) a significant difference of 0.90 (0.81; 0.98) between the pulsed and the continuous irradiation modes. A similar tendency was also determined for the corresponding RBE values relative to 70 kV X rays. Since the different findings for the comparisons of individual chromosome aberration types and combined comparisons could be explained by different sample sizes with the consequence that the individual comparisons had less statistical power to identify a difference, it can be concluded that 20 MeV protons may be slightly less effective in the pulsed mode.     

Cytogenetic effect of the pulse reactor BARS-6 continuous or one-time irradiation with ultrahigh dose rate on human lymphocytes

The results of the comparative study of radiation effects of the pulse reactor BARS-6 either in single pulse or continuos irradiation mode on human G0 lymphocytes are presented. Under identical doses the cytogenetic efficiency was observed to be higher for continuous irradiation (1 hour) than for single pulse irradiation with ultrahigh dose rate (0.5-3) x 106 Gy/minutes (pulse duration 65 x 10(-6) s). The difference averaged about 37% on total aberration frequency and 27% on the sum of dicentrics and centric rings. The influence of the dose rate and of the mixed gamma-neutron irradiation on the obtained results is discussed.     

Chromosomal radiosensitivity of human breast carcinoma cells and blood lymphocytes following photon and proton exposures

Breast carcinomas (BC) are among the most frequent cancers in women. Studies on radiosensitivity and ionizing radiation response of BC cells are scarce and mainly focused on intrinsic molecular mechanisms but do not include clinically relevant features as chromosomal rearrangements important for radiotherapy. The main purpose of this study was to compare the ionizing radiation response and efficiency of repair mechanisms of human breast carcinoma cells (Cal 51) and peripheral blood lymphocytes (PBL) for different doses and radiation qualities (⁶⁰Co γ-rays, 150 MeV and spread-out Bragg peak (SOBP) proton beams). The radiation response functions obtained using the conventional metaphase assay and premature chromosome condensation (PCC) technique enabled us to determine the number of chromosomal breaks at different time after irradiation. Both cytogenetic assays used confirmed the higher biological radiosensitivity for proton beams in tumor cells compared to PBL, corresponding to higher values of the linear LQ parameter α. additionally, the ratio of the LQ parameters β/α describing efficiency of the repair mechanisms, obtained for chromosome aberrations, showed higher numbers for PBL than for Cal 51 for all exposures. Similar results were observed for the ratio of PCC breaks determined directly after irradiation to that obtained 12 h later. This parameter (t0/t12) showed faster decrease of the repair efficiency with increasing LET value for Cal 51 cells. This finding supports the use of the proton therapy for breast cancer patients.

     

Chromosome aberration frequencies and chromosome instability in mice after long-term exposure to low-dose-rate gamma-irradiation

Chronological changes of chromosome aberration rates related to accumulated doses in chronically exposed humans and animals at a low-dose-rate have not been well studied. C3H female specific pathogen-free mice (8 weeks of age) were chronically irradiated. Chromosome aberration rate in mouse splenocytes after long-term exposure to low-dose-rate (LDR) gamma-rays was serially determined by conventional Giemsa method. Incidence of dicentrics and centric rings increased almost linearly up to 8000 mGy following irradiation for about 400 days at a LDR of 20 mGy/day. Clear dose-rate effects were observed in the chromosome aberration frequencies between dose rates of 20 mGy/day and 200 Gy/day. Furthermore, the frequencies of complex aberrations increased as accumulated doses increased in LDR irradiation. This trend was also observed for the incidences of micronuclei and trisomies of chromosomes 5, 13 and 18 in splenocytes, detected by micronucleus assay and metaphase fluorescence in situ hybridization (FISH) method, respectively. Incidences of 2-4 micronuclei and trisomy increased in mouse splenocytes after irradiation of 8000 mGy at a LDR of 20 mGy/day. These complex chromosome aberrations and numerical chromosome aberrations seem to be induced indirectly after radiation exposure and thus the results indicate that continuous gamma-ray irradiation for 400 days at LDR of 20 mGy/day induced chromosomal instability in mice. These results are important to evaluate the biological effects of long-term exposure to LDR radiation in humans.     

Polymer chromosome models and Monte Carlo simulations of radiation breaking DNA

MOTIVATION: Chromatin breakage by ionizing radiation is relevant to studies of carcinogenesis, tumor radiotherapy, biodosimetry and molecular biology. This article focuses on computer analysis of chromosome irradiation in mammlian cells. METHODS: Polymer physics and Monte Carlo numerical methods are used to develop a coarse-grained computational approach. Chromatin is modeled as a random walk on a cubic lattice, and the radiation tracks hitting the chromatin are modeled as straight lines hitting lattice sites. Each track can make a cluster of DSBs on a chromosome. RESULTS: The results obtained replace conjectured DNA fragment-size distribution functions in the recently developed RLC formalism by more mechanistically motivated distributions. The discrete lattice algorithm reproduces features of current radiation experiments relevant to chromatin on large scales. It approximates the continuous formalism and experimental data with adequate precision. It was also found that assuming either fixed chromatin with correlations among different clusters of DSBs or moving chromatin with no such correlations gives virtually identical numerical predictions.     

Dying of Gamma-Irradiated Escherichia coli Studied by the Use of Prophage

Loss of the biological activity of deoxyribonucleic acid in gamma-irradiated Escherichia coli cells was studied. The study is based on two sets of experimental data: (i) post-irradiation heat inducibility of the cells whose chromosomes were "labeled" with the thermoinducible lambdacI857ind prophage, and (ii) post-irradiation capacity of nonlysogenic cells to promote growth of the unirradiated lambdacI857ind phage. The results show that, at the beginning of incubation after irradiation, the number of plaques formed upon heat induction of lysogenic cells was much higher than the viable cell count of the nonheated culture. This high resistance of the heat inducibility gradually decreased during post-irradiation incubation. Finally, after a period of 4 h, there was no difference in sensitivity between the heat inducibility and the colony-forming ability of gamma-irradiated cells. The capacity of gamma-irradiated bacteria to support growth of unirradiated lambdacI857ind is radioresistant; this resistance, in contrast to that of heat inducibility, is much less affected during post-irradiation incubation. A continuous decrease in radioresistance of heat inducibility without a corresponding decrease in radioresistance of the capacity suggests that functional failure of initially undamaged and/or repaired parts of the chromosome gradually develops after irradiation. From the fact that after 4 h all colony formers are capable of being induced by heat, whereas no chromosomal activity can be detected in nonviable cells, two conclusions may be drawn: (i) gamma-irradiated E. coli cells destined to die reach their biological end point within 4 h of post-irradiation incubation; (ii) in most cells, functional failure of the whole chromosome is the immediate cause of death.     

Study of external low irradiation dose effects on induction of chromosome aberrations in Pisum sativum root tip meristem

The effects of low doses of ionizing radiation have been a matter of important debate over the last few years. The point of discussion concerns the validity of the linear dose-response extrapolation for low doses, used by international organizations, to establish radio-protection norms. Here, we contributed to this discussion by investigating the induction of chromosome aberrations by low to moderate doses ranging from 0 to 10 Gy in root meristem cells of 6-day-old Pisum plantlets. After acute irradiation of plantlets by a (60)Co source, the percentage of root tip meristem cells displaying chromosome aberrations was estimated immediately after irradiation and after 20 h recovery time. The dose-effect curves show non-linear responses, especially in the low dose range (0- 1 Gy), which is of particular interest. After 20 h of recovery, a steep increase of aberrations was observed for cells exposed to 0.4 Gy, followed by a plateau for doses until 1 Gy. There was an irradiation effect on plant growth during the first and second generations, showing the persistence of cell division anomalies as a long term effect of acute irradiation. This result suggests the induction of a genomic instability.Our results, in agreement with some obtained in animals, show rather non-linear dose-effect responses, with notably higher biological effects of low doses than expected.     

An analysis of the cytogenetic effects of gamma and neutron irradiation in a human lymphocyte culture at the G0 and G1 stages of the mitotic cycle (a structural-functional approach)

A study was made of the dose dependence of the chromosome aberration frequency in human lymphocytes exposed to 60Co-gamma radiation and neutrons (mean energy of 0.85 MeV) at the G0 stage and in different periods of the G1 and G1/S stages of the cycle. With gamma irradiation the dose dependence for cells at the G1 and G1/S stages was at a higher level than that for cells at the G0 stage, whereas the opposite picture was observed for cells exposed to neutron radiation. The difference was also noted in the time-response curves where gamma radiation increased and neutrons, on the contrary, decreased the aberration yield in the cells that passed from G0 to G1 stage. The experimental data obtained are attributed to activation of repair system at the G1 stage which is mainly conditioned by chromatin decondensation; the activating, that is, the functional factor influences the aberration induction with gamma irradiation, while the decondensation, that is, the structural factor, with neutron irradiation.     

Suppression of thymic lymphoma induction by life-long low-dose-rate irradiation accompanied by immune activation in C57BL/6 mice

The induction of thymic lymphomas by whole-body X irradiation with four doses of 1.8 Gy (total dose: 7.2 Gy) in C57BL/6 mice was suppressed from a high frequency (90%) to 63% by preirradiation with 0.075 Gy X rays given 6 h before each 1.8-Gy irradiation. This level was further suppressed to 43% by continuous whole-body irradiation with 137Cs gamma rays at a low dose rate of 1.2 mGy/h for 450 days, starting 35 days before the challenging irradiation. Continuous irradiation at 1.2 mGy/h resulting in a total dose of 7.2 Gy over 258 days yielded no thymic lymphomas, indicating that this low-dose-rate radiation does not induce these tumors. Further continuous irradiation up to 450 days (total dose: 12.6 Gy) produced no tumors. Continuously irradiated mice showed no loss of hair and a greater body weight than unirradiated controls. Immune activities of the mice, as measured by the numbers of CD4+ T cells, CD40+ B cells, and antibody-producing cells in the spleen after immunization with sheep red blood cells, were significantly increased by continuous 1.2-mGy/h irradiation alone. These results indicate the presence of an adaptive response in tumor induction, the involvement of radiation-induced immune activation in tumor suppression, and a large dose and dose-rate effectiveness factor (DDREF) for tumor induction with extremely low-dose-rate radiation.     

Biological indication and dosimetry of unstable chromosome aberration frequencies in human lymphocytes

Review is devoted to the problems of biological (cytogenetic) dosimetry and indication of degree of radiation lesions based on analysis of unstable chromosome aberrations in lymphocytes of human peripheral blood. Effects of radiation in low doses on human chromosomes and methodology of interpretation of the character of dose cytogenetic curves are discussed. Traditional cytogenetic analysis remains the basic one for monitoring in groups of people with accidental irradiation.     

Cytogenetic effects of UV laser radiation with wavelengths of 248, 223 and 193 nm on mammalian cells

Ten hours after irradiation of mouse cornea with doses of 0.09 to 1.5 J/cm2 the incidence of cells with chromosome aberrations increased linearly with dose and amounted to 11.7% at 248 nm, 5.5% at 223 nm and 2.6% at 193 nm per 1 J/cm2. No induced chromosome aberrations occurred 72 hr following irradiation. Within the dose range from 3.0 to 18 J/cm2 the cytogenetic effect of radiation was less manifest than that with the doses mentioned above, the frequency of chromosome aberrations being independent of either wave length or radiation dose and amounted to 2.5 to 3.0%.     

Experimental derivation of relative biological effectiveness of A-bomb neutrons in Hiroshima and Nagasaki and implications for risk assessment

Epidemiological data on the health effects of A-bomb radiation in Hiroshima and Nagasaki provide the framework for setting limits for radiation risk and radiological protection. However, uncertainty remains in the equivalent dose, because it is generally believed that direct derivation of the relative biological effectiveness (RBE) of neutrons from the epidemiological data on the survivors is difficult. To solve this problem, an alternative approach has been taken. The RBE of polyenergetic neutrons was determined for chromosome aberration formation in human lymphocytes irradiated in vitro, compared with published data for tumor induction in experimental animals, and validated using epidemiological data from A-bomb survivors. The RBE of fission neutrons was dependent on dose but was independent of the energy spectrum. The same RBE regimen was observed for lymphocyte chromosome aberrations and tumors in mice and rats. Used as a weighting factor for A-bomb survivors, this RBE system was superior in eliminating the city difference in chromosome aberration frequencies and cancer mortality. The revision of the equivalent dose of A-bomb radiation using DS02 weighted by this RBE system reduces the cancer risk by a factor of 0.7 compared with the current estimates using DS86, with neutrons weighted by a constant RBE of 10.     

Chromosome aberration frequencies produced by a 70-MeV proton beam

The effectiveness of a 70-MeV proton beam in the induction of chromosome aberrations was studied. We employed peripheral lymphocytes and analyzed the frequencies of dicentrics and rings after irradiation at doses ranging from 0.1 to 8.0 Gy at various depths within a Lucite phantom. The frequency of chromosome aberrations after irradiation with an unmodulated proton beam at 5 mm showed a dose-response relationship similar to that of 60Co gamma rays. However, irradiation at greater depths with the spread-out Bragg peak induced higher aberration frequencies at doses lower than those with gamma rays. Furthermore, the distribution curve of chromosome aberration frequencies as a function of depth was found to be slightly different from the physically measured depth-dose curve. With the spread-out Bragg peak the biological effects were more marked at greater depths, resulting in a distribution of relative biological effectiveness values. The results obtained from chromosome aberration analysis may not be related directly to those for the relationship between dose and cell killing. Slight differences in values for relative biological effectiveness due to the change of dose and site of proton beam irradiation may not be important for practical proton beam therapy, but may be important in the prevention of late radiation injuries.