The radiobiological effects of accelerated ions with high charge and high energy (HZE) on mammalian cells and their propagation in time are still not sufficiently explained and attract great deal of attention. This work aims to compare the immediate and delayed effects with emphasis on the latter. As shown by our group, the dependence of mutant fraction on expression time after irradiation may have interesting, non-monotonic, character depending on LET (linear energy transfer) of the used heavy ions. We speculate that this phenomenon may occur due to the induced genomic instability. Another area of our research is the study of the DNA structural changes in these mutants induced at different expression times. Chinese hamster V79 cells were irradiated with accelerated ions 11 B, 18 O, 20 Ne, and gamma radiation. The LET was ranging from 0.23 keV/μm of 60 Co gamma rays up to 136 keV/μm of 20 Ne ions. DNA of unique HPRT mutants was isolated, concentration measured, HPRT exons amplified, and analyzed at several different time points, up to about 40 days, after exposure. Over 1200 HPRT mutants were analyzed for deletions of exons and sorted into three main categories: partial deletion, PD—with deletion of one to eight exons; total deletions, TD—with all nine exons deleted; and no deletions—no change in the HPRT structure observed. In general, the number of samples with partial deletion was increasing with LET of the used radiation, suggesting that higher energy deposition to the cell nucleus is more likely to cause larger structural changes. In the case of total deletions, increase in their number with LET was observed up to LET ∼115 keV/μm followed by a sharp decrease. The samples were also analyzed for the distribution of deletions, in particular exons at various expression times, the so-called mutational patterns. Hypothesis of the mechanisms behind observed phenomena is given, and possible implications for further research are discussed.

The mutagenic effect of accelerated heavy charged particles on the Chinese hamster V79 cell line is studied. The induction of HPRT mutations in irradiated cells for a long expression time (up to 45 days) after exposure to radiation with different LET are analyzed. The maximum yield of mutant clones is observed at different expression times, depending on the characteristics of ionizing radiation; in this case, the position of the maximum is shifted toward later time intervals with increasing LET of radiation.

Fundamental research on the harmful effects of ionizing radiation on living cells continues to be of great interest. Recently, priority has been given to the study of high-charge and high-energy (HZE) ions that comprise a substantial part of the galactic cosmic ray (GCR) spectra that would be encountered during long-term space flights. Moreover, predictions of the delayed genetic effects of high linear energy transfer (LET) exposure is becoming more important as heavy ion therapy use is increasing. This work focuses mainly on the basic research on the delayed effects of HZE ions on V79 Chinese hamster cells, with emphasis on the induction of HPRT mutations after prolonged expression times (ET). The research was conducted under various irradiation conditions with accelerated ions 18O (E = 35.2 MeV/n), 20Ne (E = 47.7 MeV/n and 51.8 MeV/n), and 11B (E = 32.4 MeV/n), with LET in the range from 49 to 149 keV/μm and with 60Co γ-rays. The HPRT mutant fractions (MF) were detected in irradiated cells in regular intervals during every cell culture recultivation (every 3 days) up to approximately 40 days (70–80 generations) after irradiation. The MF maximum was reached at different ET depending on ionizing radiation characteristics. The position of the maximum was shifting towards longer ET with increasing LET. We speculate that the delayed mutations are created de novo and that they are the manifestation of genomic instability. Although the exact mechanisms involved in genomic instability initiation are yet to be identified, we hypothesize that differences in induction of delayed mutations by radiations with various LET values are related to variations in energy deposition along the particle track. A dose dependence of mutation yield is discussed as well.

The impact of γ radiation of 137 Сs (doses of 1 and 3 Gy), low-intensity laser radiation (λ = 670 nm, 5.3 or 10.6 J/cm 2 ) as well as the influence of consecutive laser and γ radiation on peripheral blood and blood cells (erythrocytes, leukocytes, lymphocytes, granulocytes) were studied by analyzing the number of blood cells, blood absorption spectra, and activity of antioxidant defense enzymes. Two series of experiments were performed on four groups of rats. The rats of the control group (group 1) were not exposed to γ or laser radiation. In the experimental groups, single irradiation of the whole body of rats with γ radiation (group 2), three- or four-day over-vein irradiation of blood in the tail vein by low-intensity laser radiation (group 3), and successive three- or four-day irradiation of blood by laser and then a single irradiation of the whole body with γ radiation (group 4) were performed. It was shown that changes of the blood cell content in the experimental groups are accompanied by changes in the spectral characteristics of the blood and the activity of antioxidant defense enzymes. The radioprotective effect of low-intensity laser radiation is manifested as an increase in the average number of leukocytes and lymphocytes in the group as compared with the postradiation, as well as an increase in the activity of antioxidant protection enzymes. The possibility of using low-intensity optical radiation for correction of hematological disorders caused by ionizing radiation is discussed.

The incidence of unstable chromosome aberrations in peripheral blood lymphocytes from unirradiated control subjects was analyzed using cytogenetic data obtained from 9 cytogenetic laboratories located in Moscow, St.-Petersburg, Obninsk, and Dubna (Russia). The objective of this study was to estimate the level and spectrum of spontaneous chromosome aberrations in human lymphocytes. 1140 blood samples were taken from 1112 subjects (594 men and 546 women) aged 1 to 72. The total metaphase number was 466795. The uniform Giemsa method for peripheral blood lymphocyte cultures was used. After counting 466795 metaphases, 4288 chromosomal aberrations of various types were classified. The most frequent types of aberrations were acentrics and chromatid deletions. They made up 90% of the total number of aberrations. The remaining 10% were exchange aberrations. The number of chromosome exchanges (dicentrics and centric rings) was twice the number of chromatid exchanges. Overall, the portion ofcells with chromosomal or (and) chromatid aberrations was 0.89 +/- 0.01%; the frequency of acentrics was 0.29 +/- 0.01; the frequency of dicentrics was 0.046 +/- 0.003; the frequency of unstable chromosome aberrations was 0.35 +/- 0.01; and the frequency of chromatid aberrations was 0.57 +/- 0.01 per 100 cells.

The incidence of unstable chromosome aberrations in peripheral blood lymphocytes from unirradiated control subjects was analyzed using cytogenetic data obtained from 9 cytogenetic laboratories located in Moscow, St.-Petersburg, Obninsk, and Dubna (Russia). The objective of this study was to estimate the level and spectrum of spontaneous chromosome aberrations in human lymphocytes. 1140 blood samples were taken from 1112 subjects (594 men and 546 women) aged 1 to 72. The total metaphase number was 466795. The uniform Giemsa method for peripheral blood lymphocyte cultures was used. After counting 466795 metaphases, 4288 chromosomal aberrations of various types were classified. The most frequent types of aberrations were acentrics and chromatid deletions. They made up 90% of the total number of aberrations. The remaining 10% were exchange aberrations. The number of chromosome exchanges (dicentrics and centric rings) was twice the number of chromatid exchanges. Overall, the portion ofcells with chromosomal or (and) chromatid aberrations was 0.89 +/- 0.01%; the frequency of acentrics was 0.29 +/- 0.01; the frequency of dicentrics was 0.046 +/- 0.003; the frequency of unstable chromosome aberrations was 0.35 +/- 0.01; and the frequency of chromatid aberrations was 0.57 +/- 0.01 per 100 cells.

The effectiveness of the impact of therapeutic proton beams in human cells with respect to the criterion of formation of chromosome aberrations in human-blood lymphocytes is estimated. The physical characteristics of radiation (proton LET at the input of the object and in the region of the modified Bragg peak) and the role of the biological factor (the differences in the radiosensitivity of nondividing cells corresponding to the irradiation of normal tissues along the proton-beam path and tumor tissues) are taken into account. The relative biological effectiveness of protons is ∼1 at the beam input of the object and ∼1.2 in the Bragg peak region. Taking into account the higher radiosensitivity of dividing cells in the G 2 phase of the cell cycle, the irradiation effectiveness increases to ∼1.4.

The aim of this study was to verify if the sensitivity of human peripheral blood lymphocytes (PBL) to high LET radiation is individually variable and whether it correlates with the sensitivity to low LET radiation.

The physical characteristics of the proton beam produced by a phasotron at JINR for radiation therapy are given. Chromosome damage in cells on the model of human blood lymphocytes irradiated by the initial proton beam with an energy of 170 MeV at the entrance to the object and in the Bragg peak region is studied, which corresponds to the irradiation of surrounding tissues along the beam path and tumor tissues. High proton efficiency in the Bragg peak is shown. RBE in the Bragg peak is ∼1.25 in the dose range 1–4 Gy, while the proton efficiency of the initial beam is the same as that of standard γ radiation. Since delivering a dose to a tumor is performed by irradiating the patient from several directions (up to 7), the level of cytogenetic damage to cells of the surrounding tissues on the path of the initial beam is reduced by approximately an order of magnitude. Thus, for a dose of 3 Gy in a tumor, up to 80% of its cells are damaged, while the level of damage in the surrounding tissues does not exceed 10%. The results of investigations confirm the high efficiency of proton beams for radiation therapy.

The induction of hypoxanthine-guanine-phosphoribosyl-transferase (HPRT) mutations and the survival rate of Chinese hamster cells were investigated after exposure to accelerated He-4 and C-12 ions with varying linear energy transfer (LET) (20-360 keV/mum) and to gamma rays. The power law was found for the mutation induction rate as a function of the radiation dose with LET up to 50 keV/mum. The dependence transforms into a linear one at higher LET values. The RBE of heavy charged ions increases with the LET of ions to maximal values at 80-100 keV/mum. The RBE values for the induction of mutations and chromosomal aberrations amount to 4.8-5.0 and are twice as high as the values corresponding to the cell-survival criterion (2.3-2.9). A high heterogeneity and chromosomal instability in aneuploidy and chromosomal-aberration levels were observed in spontaneous mutants and in those induced by protons (LET of 0.218 keV/mum), N-14 ions (LET of similar to77 keV/mum), and gamma rays. The chromosomal instability was highest for spontaneous mutants and decreased with increasing LET. The metabolic hypothesis for the chromosomal instability is discussed. The regularities in the formation of unstable chromosomal aberrations in human-blood lymphocytes were studied after exposure to protons, 12C, Mg-24, and N-14 ions (LET of 0.218, similar to12, 42.7, and similar to77 keV/mum, respectively), and gamma rays. The rate of aberrations increased with the dose and radiation LET. The RBE values were 1.0. 1.2-1.3, 1.4-1.7, and 2.0-2.2 for protons, C-12, Mg-24, and N-14 ions, respectively. The FISH analysis of stable chromosomal aberrations (translocations) revealed a high efficiency of N-14 ions (RBE of similar to3.0). According to all cytogenetic tests, there were no essential differences between the efficiency of protons and gamma rays. The dose dependence of the rate of production of cells with chromosomal aberrations after exposure of human lymphocytes, Chinese hamster cells, and human melanoma cells to gamma rays and their adaptive response were studied in the dose range of 0.01-1 Gy. For all types of cells, a nonlinear dose-effect dependence was revealed for cells with chromosomal aberrations. This dependence is characterized by a hypersensitivity of cells at doses of 0.01-0.2 Gy and an induced radioresistance at doses above 0.5 Gy. The possible mechanisms involved in this phenomenon are discussed.

The induction of HPRT-mutations and survival of Chinese hamster cells (line B11ii-FAF28, clone 431) were studied after irradiation by 4He and 12C-ions of various LET (20 – 360 keV/μm), produced by the U-200 heavy ion accelerator. The RBE increases with LET up to the maximum at 100–200 keV/μm and then decreases. Cytogenetic analysis was performed on the HPRT-mutant subclones selected from unirradiated Chinese hamster V-79 cells and from HPRT- mutant subclones that arose after exposure to γ-rays, 1GeV protons and 14N-ions (LET - 77 keV/μm), produced by the synchrophasotron and the U-400M heavy ion accelerator. Slow growing mutant subclones were observed. The cytogenetic properties of individual clones were highly heterogeneous and chromosome instability was observed in both spontaneous and radiation-induced mutants. Chromosome instability was highest among spontaneous mutants and decreased with increasing LET.

Formation of stable and unstable chromosome aberrations in human blood lymphocytes is investigated with the use of the FISH technique and the standard metaphase method. Lymphocytes were irradiated in vitro with accelerated protons with an energy of 1 GeV (LET similar to 0.218 keV/mum) and nitrogen ions N-14 with an energy of 50 MeV/nucleon (LET similar to 77 keV/mum), generated by synchrophasotron and U-400M JINR accelerator, respectively. Using the FISH method, a high incidence of chromosomes-1 and -2 translocations was revealed. Those aberrations ranged from 40 to 45% after irradiation with protons and gamma-rays and up to similar to25% after irradiation with nitrogen ions. A high incidence of fragmentation of these chromosomes under irradiation with nitrogen ions up to 50% of the total number of aberrations was detected; when irradiated with protons and gamma-rays, they made up similar to25%. Using both methods, we find a linear-quadratic dose-response dependence for the total number of aberrations, incidence of translocations and chromosomes-1 and -2 dicentrics, and also a linear dose-response dependence for the number of cells with aberrations together with that for the rate of formation of chromosome fragments under irradiation with protons and gamma-rays. When irradiated with nitrogen ions, the dependence on the dose is a linear one if the data on cytogenetic indices is used. It is established that the effect produced by protons with an energy of 1 GeV does not differ from that of gamma-irradiation. A high efficiency of nitrogen ions was revealed; the RBE coefficients for the translocations yield were 3.1; for other indices, they varied from 3 to 6.

PURPOSE:To detect the frequencies of interchanges among 11 chromosomes in lymphocytes irradiated with gamma-rays and to find out whether these frequencies reflect the proximity of some of these chromosomes within the interphase nucleus. MATERIAL AND METHODS:Exchange aberrations were detected in the first mitosis after irradiation of human lymphocytes with 3 and 5 Gy gamma-rays of 60Co. Two-colour repeated FISH with two differently chemically modified probes in each hybridization was applied. The microscope stage positions of each mitosis were recorded after the first hybridization and used for the automatic scanning of images after all successive experiments. Five images were obtained for each mitosis differing in visualized pairs of chromosomes. Comparing these images, exchanges among 10 chromosomes could be detected. Painting of the p arm of chromosome 21 with the painting probe for chromosome 22 also made it possible to detect exchanges of this chromosome with other chromosomes of the selected group. RESULTS:Frequencies of exchange aberrations induced in chromosomes of the selected group as well as interchanges between many pairs of chromosomes of this group were roughly proportional to the DNA content of chromosomes. Higher frequencies of interchanges than expected according to the model of linear proportionality were found between several chromosomes involved in translocations frequent in different subtypes of leukaemia. CONCLUSIONS:Frequencies of interchanges among 11 chromosomes of human lymphocytes induced by gamma-rays do not indicate as clearly as fast neutrons the non-random arrangement of chromosomes in the cell nucleus. The interaction of a large number of chromosomes in exchange aberrations suggests that the chromatin in the territory of one chromosome is accessible for several other chromosomes.

On the basis of literature and proper data the inference was made about the essential role of structural chromosomal (and gene) damages in spontaneous and radiation-induced mutagenesis of mammalian and human cells on HPRT-loci. The evidence of the increasing role of these damages in the mutagenesis after the influence of ionizing radiation with high LET are considered. The consequences of HPRT-gene damages have been examined hypothetically. The heterogeneity of mutant subclones on their cytogenetical properties were revealed experimentally. The data reflect a phenomenon of the reproductive chromosomal instability in many generations of mutant cells. The mutagenesis of mammalian cells is also accompanied by the impairment of chromosome integrity with high probability as a stage of appropriate genome reorganization because of changed vital conditions.

These recommendations focus on the action of ionizing radiation on eukaryotic chromosomes.

Base oneself on analysis of literature and proper data the conclusion was inferred about essential role of structural chromosomal (and gene) damages for radiation-induced mutagenesis of mammalian and human cells (for example HPRT loci). The evidences are adduced of their increase role in mutagenesis after influence of ionizing radiations with high LET. The consequences of HPRT-gene damages have been examined hypothetically.