The cytogenetic damage, radiosensitivity and adaptive response in children living in the different districts of Moscow

The spontaneous level of blood lymphocytes with micronuclei (MN), the sensitivity to 1.0 Gy irradiation and adaptive response (AR) after adaptive irradiation with a dose of 0.05 Gy 5 hr later have been studied in children population living in different districts of Moscow. It was shown that spontaneous frequency of cells with MN, the sensitivity to 1.0 Gy acute irradiation and the AR manifestation have significant differences in samples taken from children living in different districts. The individual variability is significant also. In each group of children the individuals with the enhanced radiosensitivity after adaptive irradiation have been observed. In conformance with the data of radioecological inspection the radiation situation in different Moscow districts is quite safe on overage but in some districts the spontaneous level of lymphocytes with MN, and radiosensitivity after 0.05 Gy irradiation were enhanced, the AR was not found.     

On the new mechanism of adaptive response

The role of changes in cell composition of population of human blood lymphocytes in the forming of an adaptive response (AR) has been studied. By micronuclei assay and cytokinetic block with cytohalasin B the frequency of mono-, bi- and multinuclear cells with micronuclei (MN) and without MN were determined in the initial population. The same parameters have been studied after exposure of the population to the adaptive (0.05 Gy), challenge (1.0 Gy) doses and to doses 0.05 + 1.0 Gy 5 hours after. 13 from 23 investigated individuals manifested the AR: the decreasing of the ratio of damaged binuclear cells to the all binuclear cells after the adaptive and challenge exposure. It was shown that the ways of an AR forming are different: in 7 of 13 individuals with AR the number of binuclear cells with MN did not decrease but the amount of binuclear undamaged cells increased. The ratio of these parameters enhances but not for the account of cells with MN decreasing. There is the linear correlation between the frequency of cells with MN and the frequency of binuclear cells in population (spontaneous, after irradiation with doses of 0.05, 1.0 and 0.05 + 1.0 Gy) with the coefficient of correlation about -1. These results show the presence of new mechanism of AR forming, which is not connect with the induction of damage repair and rather with the stimulation of cell division. In the another group of individuals the decrease in damaged cells number after irradiation with doses of 0.05 + 1.0 Gy have been observed. Probably the stimulation of repair system occurred to the moment of 1.0 Gy irradiation. Thus, the mechanism of an AR forming depends on the individual properties of organism. The work was suppoted by RFBR grant 03-04-48325a.     

Cytogenetic results of irradiation of Southern Ural residents

The paper summarizes the results of studies of 85 individuals exposed in the Southern Ural region. The spontaneous frequency of the cells with micronuclei (MN) in a population of human blood lymphocytes after PHA stimulation and cytokinetic block with cytochalasin B has been determined. The sensitivity of lymphocytes to the irradiation at the dose of 1.0 Gy and the adaptive response (AR) after the irradiation at the low adaptive dose of 0.05 Gy, and the challenge dose of 1.0 Gy 5 h later have been studied too. It was shown that the peculiarity of the Urals population consists in a higher individual variability of the frequency of cells with MN in all groups have been investigated (spontaneous, after acute irradiation in the dose 1.0 Gy) in comparison with Moscow people. The proportion of persons with a significant AR in the Urals groups was considerably lower than that identified among Moscow residents, and the number of persons with enhanced radiosensitivity increased following low-dose irradiation. We can suppose that prolonged action of low level radiation with another ecological factors, living in the contaminated regions result in the enhancement of the sensitivity to the genotoxic agents in the separate individuals.     

The mechanisms of adaptive response. Estimation of capacity of human blood lymphocytes to radiation adaptive response using different criteria

Blood lymphocytes of 15 healthy donors have been investigated for the ability to decrease their radiosensitivity after treatment with low dose irradiation named radioinduced adaptive response (AR). The unstable chromosome aberrations were used to evaluate the radiosensitivity change after irradiation of cells with low adaptive dose (5 cGy) and subsequent high challenge dose (1.0 Gy) in comparison with the effect of challenge irradiation only. Three indexes were used: the frequency of cells with aberrations in all analyzed cells (A), the number of chromosome aberrations per cell (B) and the number of chromosome aberrations per one aberrant cell (C). It has been discovered that all donors examined can be divided into four groups: 1--individuals which cells did not show AR by all indexes used; 2--individuals which cells showed AR by indexes A and B, but not C; 3--AR was demonstrated by indexes B and C; 4--AR was confirmed by all three indexes. Generally accepted repair model for AR formation explains only the case of donor groups 3 and 4, but can not explain the mechanism leading to the case of group 2. For understanding this mechanism, the distribution of metaphases by the number of chromosome aberrations per cell was analyzes for each donor. It was shown that the part of cells without aberrations in group 2 donors increased significantly after treatment with the adaptive and challenge irradiation in comparison with that after irradiation with challenge dose only. The conclusion is that in this case AR is formed as a result of change in the frequency 0 cell class--population shift. The analogous shift was observed in the distributions of metaphases for all donors of the group 4, but was absent in the group 3 donors. The data obtained suggest that AR of blood lymphocytes might be a result of several processes: activation of submutational genome damage repair; population shifts manifested by the change in the part of undamaged cells; and, possibly, activation of apoptotic cell death. The complex nature of AR affects each of radiosensitivity evaluation criteria to a different extent.     

Mechanism of adaptive response: Estimation of human lymphocyte adaptive response to radiation using various criteria

Blood lymphocytes of 15 healthy donors have been investigated for their ability to reduce radiosensitivity after low-dose irradiation-radio-induced adaptive response (AR). The frequency of unstable chromosome aberrations was used to evaluate cell radiosensitivity after the irradiation of cells in low adaptive (5 cGy) and high challenge (1 Gy) doses in comparison with the effect of challenge irradiation only. Three indexes have been used, i.e., (A) the frequency of cells with aberrations per total analyzed cell, (B) the number of chromosome aberrations per one cell, (C) and the number of chromosome aberrations per one aberrant cell. It was found that the donors can be divided in the four following groups: 1. AR was not estimated any of the indexes used; 2. AR was estimated with indexes A and B, but not C; 3. AR was shown by indexes B and C; 4. AR was evident with all three indexes. The generally accepted AR repair model only explains the appearance of group-3 and-4 donors, but not group-2. For the purpose of understanding the AR mechanisms and the difference in AR estimations with various criteria, the metaphase distribution by the number of chromosome aberrations has been analyzed for each donor. It was shown that, in group-2 donors, the number of cells without aberrations after adaptive and challenge irradiations was significantly higher than after irradiation with a challenge dose only. Thus, in this group, AR is formed as a result of the changed frequency of cells in the 0 class (population shift). A similar shift is observed in the metaphase distribution in the donors of group 4, but not in group 3. The data obtained show that AR is probably a result of several processes, including the activation of the reparation of premutational genome damages, population shifts evident in the frequency of undamaged cells, and, possibly, the activation of apoptotic cell death. The complex character of AR is reflected to different degrees in each criterion of radiosensitivity.     

An individual variability of the adaptive response to irradiation in human cells. Approach to its determination

On human blood lymphoxytes with micronuclei (MN) assay and cytokinetic cytochalasin block and analysis of chromosome aberrations the change of cell population composition, adaptive response (AR) and phenomenon of enhanced radiosensitivity after low dose (5 cGy) and challenge doses (1.0 Gy) have been studied. Irradiation have been carried out in G1 and G2 phases of cell cycle (24 h and 48 h after PHA stimulation). Fixation of cells have been conducted after 50 h (2 h after demecolcin adding) and 72 h (24 h after cytochalasin adding) chromosome and MN assay. Evaluation criteria were the frequency of binucleated cells with MN on 1000 binucleated cells and the frequency of cells with chromatid aberration on 100 metaphases. It was shown that cell population constitution change, AR occurring depended on the individual peculiarity. The evaluation of AR presence by the indexes of bimucleated cells with MN frequency and cells with chromatid aberrations don't coincide (coincidence is observed in 3 cases from 15). It is supposed that in G2 phase after irradiation in challenge dose the MN assay and metaphase analysis can register different cells (24 h and 2 h after mitotic block). The cell population constitution change can probably influence on the AR evaluation but in isn't the AR mechanism. The main mechanism of AR forming * the protection from the damages by different ways. AR depends on many factors, individual peculiarities observes by the use of definite evaluation criteria, in individuals with definite genetic constitution. Perhaps these considerations permit to discuss the problem of AR universality.     

The cell composition in the lymphocyte population and the radiation adaptive response

Using lymphocytes of 5 healthy individuals the ability to adaptive response (AR), cell composition of population after PHA stimulation, changes in cell composition population after irradiation in the dose of 1.0 Gy and after irradiation in adaptive (0.05 Gy) and challenge (1.0 Gy) doses have been studied. AR observed in 2 of the 5 individuals only. After PHA stimulation the persons with AR have the total amount of cells after mitosis or during mitosis (the number of binucleated cells + the number of multinucleated cells + the whole cells with micronuclei + the number of mitotic cells) on average is higher than in persons without AR. In individuals with AR the linear correlation between the number of binucleated cells with micronuclei (on the 1000 scored binucleated cells) and the part of binucleated cells in the population is observed with coefficients of correlation -0.89 and -0.91. In the humans without AR this correlation is absent. The correlation observed permits to suppose that AR may occur at the expense of not only the decrease in number of damaged lymphocytes, but also the increase in the share of not damaged binucleated cell with the stable number of damaged cells.     

Increased radiosensitivity after irradiation of lymphocytes low adaptive doses

The variability of blood lymphocyte reaction on the adaptive irradiation (0.05 Gy at first, then 1.0 Gy 5 h later) was investigated by micronuclei assay. Blood samples were obtained from 700 children. It was shown that in all groups studied there were children with enhanced radiosensitivity ("radiosensitivity syndrome"-RS) after exposure to adaptive low dose of radiation. The radiosensitivity syndrome occurred more often in groups of ill children; part of them was characterized by the enhanced blood content of immunoglobulin E, enhanced level of T helpers and T suppressors. A high spontaneous level of lymphocytes with micronucleus is a factor of radiosensitivity formation. The possible factors resulted in radiosensitivity syndrome are discussed.     

Reciprocal adaptive response of human peripheral lymphocytes induced by bleomycine or gamma rays.

The adaptive response and reciprocal adaptive response induced in vitro by exposure to low doses of gamma rays (0.05 Gy) or bleomycin (0.05 microg/ml) in human peripheral blood lymphocytes were assessed by the frequency of chromosome aberrations. Gamma rays (1.5 Gy) or bleomycin (1.5 microg/ml) were used as the challenge doses. In the experiments, blood samples from 5 healthy donors were investigated. It has been found that low doses of bleomycin and gamma rays induced a reciprocal adaptive response to high doses of gamma rays or bleomycin. Moreover, the results confirmed that the adaptive response did not correlate with the radiosensitivity of the peripheral blood lymphocytes.     

The adaptive response of first generation offsprings irradiated parents

In offspring's of first generation irradiated inhabitants of Techa river (fathers, mothers and both parents) the spontaneous level of damaged blood lymphocytes, sensitivity of lymphocytes to the additional acute irradiation in dose 1.0 Gy and radioinduced adaptive response after adaptive (5 cGy) and challenge (1.0 Gy) irradiation 5 h after was studied. The micronuclei test with cytochalasin B as a criteria of the effect have been used. It was shown, that descendents of irradiated parents differ from the control group. The main difference is the significant decrease of the adaptive response frequency in the progeny. In the offspring's of the irradiated fathers and mothers there is no one individuals with the adaptive response; in the offspring's of both irradiated parents the frequency of individuals with adaptive response decreases in control from 19.5% to 6.8%. The distribution of descendents according to response on adaptive irradiation differ significantly from the control distribution and from the each other. And the tendency to the radiosensitivity increase after adaptive irradiation was observed. In the whole joint group of progeny the mean spontaneous cell frequency with micronuclei decreased, but the sensitivity of lymphocytes to the additional acute irradiation doesn't differ from the control. The results of the paper permit to suppose that transgenerational genome instability in human can be determined. Earlier discovered decrease of the adaptive response frequency in the Techa river livings is observed in the offspring's of irradiated fathers, mothers and both parents.     

Molecular-biological properties of blood lymphocytes of Hodgkin's lymphoma patients. Plausible possibility of treatment effect prognosis

Hodgkin's lymphoma (HL) patients were investigated before and during chemical and radiation therapy. The properties of peripheral blood lymphocytes of the HL patients before treatment have been compared with healthy donors and the patients during the treatment. The genetic damage--frequency of cells with micronuclei (MN), the level of DNA single- and double-strand breaks (SSB and DSB), DNA-protein cross-links (DPC) have been studied. Biochemical and physiological parameters have been compared as well: the concentration of reactive oxygen species (ROS), the ability to the adaptive response induction. The radiosensitivity of lymphocytes in vitro exposed to the 1 Gy irradiation has also been determined (by MN test). It was shown that in Hodgkin's lymphoma patients' lymphocytes (in comparison with healthy donors) the frequency of cells with MN does not change, the level of SSBs and DSBs increases, the amount of DPC does not change, and ROS concentration (on average) significantly increases because of the part of the population that have high ROS content. The ROS concentration decreases to control level, the frequency of cells with MN increases, the level of DSBs does not change but the level of DPCs (which prevents the determination of DSB) increases in the patients during treatment. It was also discovered that lymphocyte radiosensitivity correlates with the MN cells frequency before treatment and the ROS concentration. These results make it possible to suppose that the high MN frequency and high ROS concentration in Hodgkin's lymphoma patient lymphocytes (before treatment) can serve as prognostic factors for the effectiveness of radio and chemical therapy.     

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).     

Adaptive response in irradiated human lymphocytes: radiobiological and genetical aspects

The adaptive response (AR) in human lymphocytes in different experimental protocols was investigated. The AR was found to be present in cells pre-exposed to 3 cGy of X-rays in G0, G1 and S phase as well as with tritiated water (4 muCi/ml) when the 'challenge' dose was given in G2. There was no AR after prior exposure of the cells in S phase to secondary irradiation from 70 GeV protons. The AR was not observed after preliminary X-irradiation of the lymphocytes in G0 and G1 and 'challenge' irradiation in G1. Cells from 6 patients with Down's syndrome were tested. At least 5 of them did not show the AR. The AR is considered to be a phenomenon of the antimutagenic aftereffect.     

Effect of low dose irradiation on the reaction of blood lymphocytes of individuals with the somatic diseases

The reaction of blood lymphocytes on adaptive irradiation in vitro (0.05 Gy) and challenge irradiation (1.0 Gy) 5 h after has been studied among 320 children 3-16 years old by micronuclei test with cytochalasin B cytokinetic block. The adaptive response or the hypersensitivity phenomenon (HS) has been determined by the calculation of the number of binucleated cells with micronuclei (MN) in 1000-2000 binucleated cells. For each individual by the chi2 criteria significant differences between the frequencies of damaged cells by irradiation in challenge dose and by combined action of adaptive and challenge doses have been detected. On the base of the results obtained the connection between AR or HS and the somatic diseases of different type and the disturbancies in systems of humoral and cell immunity has been studied. It was shown that in the row of allergic diseases and tuberculous infection among the significant part of the children (approximately 50%) the HS phenomenon was registered. The connection between the HS phenomenon and inflammatory processes (without allergic components), the logopedic disturbancies wasn't observed. It was shown in the group of HS children the deviation in the cell immunity system is registered. Significant deviations in HS group in the system of the humoral immunity is observed. So by the HS phenomenon significant number of the children have the somatic diseases and the deviation in the systems of cell and humoral immunity.     

Cytogenetic and survival adaptive responses in G1 phase human lymphocytes.

Human lymphocytes from six donors were treated with 5 cGy of X-rays followed by 200 or 400 cGy in the first G1 phase after PHA stimulation, and assayed for cytogenetic aberrations and cell survival. Four donors showed cytogenetic adaptive responses with 400 cGy, and one with both 200 cGy and 400 cGy. Both exchanges and deletions were reduced, indicating that the cytogenetic adaptive response acts by restitution of chromosome breaks. Good correlations were found between nonaberrant cells and survival, although the former were often higher than the later, especially with the 400 cGy dose. In four of six donors, 5 cGy alone had significant effects on cell survival; however, this was independent of the 5 cGy effect on high-dose-induced responses. Two donors had survival adaptive responses with 5 + 200 cGy, while none were found with the 5 + 400 cGy treatment. The comparisons between the cytogenetic and survival responses suggests that a survival adaptive response will only be seen with a sufficient increase in nonaberrant cells. To date, adaptive responses to ionizing radiation have been reported to occur in G0, G1 and late S/early G2 human lymphocytes.     

Protective effect of ferulic acid on gamma-radiation-induced micronuclei, dicentric aberration and lipid peroxidation in human lymphocytes

In this study we examined radioprotective effect of ferulic acid (FA) on gamma radiation-induced dicentric aberration and lipid peroxidation with reference to alterations in cellular antioxidant status in cultured lymphocytes. To establish most effective protective support we used three different concentrations of FA (1, 5 and 10 microg/ml) and three different doses of gamma-radiation (1, 2 and 4 Gy). Treatment of lymphocytes with FA alone (at 10 microg/ml) gave no significant change in micronuclei (MN), dicentric aberration (DC), thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) or glutathione peroxidase (GPx) activities when compared with normal lymphocytes; irradiation at 1, 2 and 4 Gy increased the MN and DC frequencies in a dose-dependent manner. Treatment with FA for 30 min before radiation exposure resulted in a significant decline of MN and DC yields as FA concentration increased. Compared to 1 Gy exposure alone, the extent to which FA (1 microg/ml) reduced the MN and DC yields was 75% and 50%, respectively. With 4 Gy irradiation, FA (10 microg/ml) decreased 45% MN and 25% DC frequencies. FA-pretreated lymphocytes (1, 5 and 10 microg/ml) showed progressively decreased TBARS levels after irradiation. Irradiation (1, 2 and 4 Gy) significantly decreased GSH levels, SOD, CAT and GPx activities in a dose-dependent manner. Pretreatment with 10 microg/ml of FA significantly (p<0.05) prevented the decreases in the radiation-induced GSH, SOD, CAT and GPx activities. These findings suggest potential use and benefit of FA as a radioprotector.     

The reaction of cell population to low level of irradiation

The results of long-term investigations of the effectivity of low level irradiation (below 0.5 Gy) carried out on the cells in culture and blood human lymphocytes (adults and children) have been brought. In the experiments conducted in the laboratory conditions and in the contaminated with radionuclides regions (after Chernobyl accident) the genomic instability have been discovered. The cell manifestations of the genomic instability have been registered in the progeny of irradiated cells as the decreasing of proliferative activity, the increasing of the frequency of cells with micronuclei, the increasing of cells with sister chromatid exchanges, the late cell death, the absence of the adaptive response ability, the enhancement of the radiosensitivity. The results of the investigations of the adaptive response of blood lymphocytes have been presented. It was shown that in all populations investigated there are individuals without the adaptive response and the individuals with the enhancement of radiosensitivity after adaptive irradiation (0.05 Gy). On the basis of own results and the data of literature the possible mechanisms of low level irradiation effects are discussed. The conclusion is that: a. The population with new properties can be formed after low level irradiation; b. The effects and mechanisms of this effect realization can be different from that after irradiation with high doses.     

The dose dependence of cytogenetic damages and the adaptive response of mammalian cells under the action of ionizing radiation at low doses

The dose-effect dependence of cytogenetic damage after single dose irradiation in the dose range of 0.1-2 Gy and the adaptive response after double-dose irradiation were studied on Chinese hamster and human melanoma cells in culture. The non-linear dose dependencies were found for the induction of chromosome aberrations with decrease in cell radiosensitivity in the definite dose range. This decrease started at 10 and 20 cGy for melanoma and Chinese hamster cells respectively. The maximal adaptive response was induced at 1 cGy for melanoma cells and at 20 cGy for Chinese hamster cells. It can be supposed that the same inducible repair processes are responsible for non-linearity of dose-effect curves and induction of the adaptive response. These processes are similar in mechanisms and different in quantitative proportion for different cell types.     

Frequency of chromosome aberrations induced in human peripheral lymphocytes by in vitro 60Co gamma quanta at doses of 1 to 5 Gr. in an analysis of the 1st-division cells at different fixation times

The quantitative analysis of chromosomal aberrations in the first division cells of 50-, 54-, 58-, 52- and 66-hour peripheral blood lymphocytes cultures of healthy donors was performed after irradiation in vitro with 60Co gamma-quantums at doses 1--5 Gy. Cells of the first division were identified by a differential staining of sister chromatid method using 5-bromdeoxyuridine. No significant differences in frequencies of aberrant cells and aberrations of chromosomal type were found between cultures fixed at different times. The distribution of dicentrics in cells did not differ from the Poisson distribution regardless of fixation times and doses. On the basis of these findings it is concluded that chromosomes of human peripheral blood lymphocytes passing the cell cycle at different rates have approximately equal radiosensitivity.     

The cytokinesis-block micronucleus assay as a biological dosimeter in spleen and peripheral blood lymphocytes of the mouse following acute whole-body irradiation

To evaluate the application of the cytokinesis-block (CB) micronucleus (MN) assay as a biological dosimeter following in vivo exposure to ionising radiation we determined the micronucleus frequency in spleen and peripheral blood lymphocytes of the mouse, serially, for 14 days following acute whole-body irradiation. The baseline MN frequency of spleen lymphocytes (7.86 +/- 0.68, mean +/- 1 SD) was significantly (p less than 0.001) elevated when compared to that for peripheral blood lymphocytes (4.10 +/- 0.53). Immediately after irradiation there was a substantial dose-related increase in MN, but the MN frequencies in spleen lymphocytes (120.2 +/- 9.4 for 1 Gy; 409.5 +/- 38.4 for 2 Gy) were significantly (p less than 0.009) elevated compared to those in peripheral blood lymphocytes (78.0 +/- 7.0 for 1 Gy; 200.2 +/- 10.9 for 2 Gy). During the 14 days after irradiation, the MN frequency in spleen lymphocytes declined gradually to approximately half of the value observed immediately after irradiation. By contrast the MN frequency in peripheral blood lymphocytes increased during the week after irradiation, but ultimately MN frequencies in blood and spleen became approximately the same by day 14. Study of isolated murine lymphocytes irradiated in vitro showed that the number of MN generated by a given dose of radiation was approximately 2-3 times greater than the number generated by in vivo irradiation. These results suggest that measurement of MN in vivo after irradiation can be used as an in vivo dosimeter. However, precise dosimetry is probably affected by factors such as kinetic changes in different lymphocyte populations and possibly by in vivo factors which influence sensitivity of cells to radiation.