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 response of a cell population to low-dose irradiation

The cell composition of a population of human blood lymphocytes was studied after irradiation at doses of 5 cGy, 1.0 Gy and 5 cGy + 1.0 Gy and the use of a cytokinesis block. The frequencies of uni-, bi- and multinucleate lymphocytes with and without micronuclei (MN) were taken into account. By the standard criterion the frequency of binucleate lymphocytes with MN among binucleate lymphocytes--the donors were characterized as follows: in with reduction of radiosensitivity after irradiation with 5 cGy + 1.0 Gy as compared to the values of radiosensitivity after irradiation with 1.0 Gy only (an adaptive response, AR); in with no change of radiosensitivity after exposure to these doses (no AR); and with an increased ofradiosensitivity after exposure to these doses (syndrome of increased radiosensitivity, IRS). It was found that upon exposure to 1.0 Gy and 5 cGy + 1.0 Gy in some donors with AR, without AR and with IRS the total numbers of damaged cells in the population and the number of binucleate cells with MN were equal. This result calls in question the involvement of the repair mechanism in the alteration of radiosensitivity of lymphocytes in these donors. It was also observed that in the same donors a simultaneous increase (or a decrease in the case of IRS) of the portion of undamaged binucleate cells in the population took place. Our results demonstrate the existence of a new, populational, mechanism involved in the alteration of radiosensitivity after exposure to the adaptive and challenge doses.     

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 molecular and cellular consequences of the chernobyl accident

In this paper the results of research at 5–10 and 24 years after the Chernobyl accident are summarized. These results include the investigation of genomic instability, formation of the adaptive response, genome damage, and oxidative status. The studies were performed on cells in culture, mice, children and adults who lived in the contaminated areas, and liquidators of the consequences of the Chernobyl accident. Inhibition of cell proliferative activity, late cell death, and the increase in micronucleus and giant cell frequency were observed after the exposure of cells in culture in the accident zone followed by their culturing in laboratory conditions. In the progeny of the exposed cells, the effect of enhanced radiosensitivity was detected. Thus, it can be assumed that exposure of parental cells in culture in the area of the accident induced genomic instability that resulted in the development of various abnormalities in progeny cells. At the organism level, the Chernobyl zone exposure of mice caused an increase in radiosensitivity; as well, a decrease in the endotheliocyte density in the cerebral cortex and other brain tissues was observed. In the blood lymphocytes of children stimulated by PHA, a more than two times increase in micronucleus cell frequency was detected. A reduced number of individuals with significant adaptive response was found in both the juvenile and adult groups. In all investigated populations, an increased number of individuals with enhanced radiosensitivity were observed in response to low-dose radiation exposure. At 24 years after the accident liquidators were subjected to examinations, which revealed an increased frequency of cells with micronuclei and chromosome-type aberrations in blood lymphocytes, an elevated level of DNA double strand breaks, and a reduced level of reactive oxygen species compared to those of the control group. This means that the genomic instability that was accumulated by the residents of the contaminated regions and liquidators as a result of the accident leads to damage of the genetic apparatus, an increase in radiosensitivity, and hypoxia as late consequences that all are risk factors and increase the probability of the development of tumor and non-tumor diseases. The development of the above-mentioned pathological processes may occur in the distant future.

     

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.     

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.     

Detection of genomic instability in offspring of male mice chronically exposed to gamma-radiation by the "adaptive response" test

The genomic instability (GI) in somatic cells of the progeny (F1 generation) of male mice chronically exposed to low-dose gamma-radiation was studied by comparative analysis of chromosome damage. BALB/C male mice exposed to 0.1 Gy (0.01 Gy/day) and 0.5 Gy (0.01 and 0.05 Gy/day) were mated with unirradiated females 15 days after irradiation. For comparison of radiosensitivity, two-month-old males, the descendants of irradiated and unirradiated animals, were subjected to irradiation with a dose of 1.5 Gy (0.47 Gy/min) from a 60Co source. GI was revealed by the standard scheme of adaptive response. The experiments indicated that, by using the test "adaptive response", it is possible to detect the transition of gamma-radiation-induced genomic instability in sex cells of male parent into somatic cells of mice (F1 generation) either from changes in radiosensitivity or by the absence of the adaptive response induced by a standard scheme.     

Is interindividual variation of cellular radiosensitivity real or artifactual?

A recently developed dose-survival assay using human G0 T lymphocytes from peripheral blood was employed to assess possible interindividual variation of cellular radiosensitivity by comparing variability between a single test for different individuals and repeated tests for a single donor. The surviving fraction at each X-ray dose level fluctuated similarly between the two groups, and the X-ray dose required to kill 90% of the cells (D10) was 3.59 +/- 0.18 Gy (mean +/- SD) for 31 different individuals and 3.66 +/- 0.21 Gy for 28 repeated tests of one individual. Analysis of variance to compare the two sets of data showed that variation in the D10 value was not significantly greater in the former group. Analysis of D50 and D90 showed similar results. These results support the hypothesis that interindividual variation in cellular radiosensitivity is quite small, if it exists at all, as far as can be determined by the loss of colony-forming ability of irradiated G0 lymphocytes.     

The effect of low-dose irradiation and of age on the in vitro radiosensitivity of human lymphocytes

The effect of low-dose irradiation and of age on the radiosensitivity of human lymphocytes was studies in two groups: control (67 people) and exposed to uncontrolled low-dose irradiation in past (165 people). Radiosensitivity of lymphocytes was estimated by the level of chromosome aberrations induced in vitro by gamma-radiation Cs137 at the dose 1.5 Gy. In exposed children the frequency of induced chromosome aberrations was higher and in the exposed adults--lower in comparison to the coresponding controls. To investigate an age response of the number of chromosome aberrations three statistical approaches were used: the correlation analysis of individual data, the correlation analysis of means for 10-years intervals, the comparison of 3 age groups. In control group no significant alteration in the level of induced chromosome aberrations with age was found. However the significant negative correlation between these two parameters was revealed in exposed group, which likely is due to the opposite direction of differences in radiosensitivity of exposed children and adults from the corresponding controls.     

Molecular and cellular consequences of the Chernobyl accident

In this paper the results of the Chernobyl accident investigation 5-10 and 24 years after are summarized. The genomic instability, adaptive response formation, genome damage and oxidative status have been investigated. The studies were performed on cells in culture, mice, children and adults living in contaminated areas and liquidators. On cells in culture after exposition in the accident zone and culturing thereafter in laboratory conditions the cell proliferative activity decrease; the late cell death, the frequency of cells with micronuclei and giant cells increasing have been observed. In the progeny of exposed cells the enhancement of radiosensitivity has been noticed. So we can suppose that in cultured cells exposition in the zone of the accident the genomic instability is induced which results in many disturbances. At the organism level in mice exposed in the Chernobyl zone the radiosensitivity increase and the decrease of endotheliocytes density in brain tissue has been observed. On the stimulated by PHA blood lymphocytes of children the increase of the frequency of cells with micronuclei more than 2 time have been noticed. In all groups investigated, the decrease of individuals with significant adaptive response was observed. In children and adults inhabitants the increase of radiosensitivity after low dose of irradiation has been noticed. 24-year after the accident it was discovered that in liquidators lymphocytes the frequency of cells with micronuclei, with chromosome type aberrations, with DNA double strand breaks have been increased; the reactive oxygen species (ROS) were decreased in comparison with the control population. We can suppose that genomic instability induced in residents of contaminated regions and liquidators long after the accident results in the genetic apparatus damage, radiosensitivity enhancement, hypoxia that represent risk factors and increase the probability of tumour and non-tumour diseases. The development of these pathological processes may happen in much more remote periods.     

Radiosensitivity of CD4 or CD8 positive human T-lymphocytes by an in vitro colony formation assay

The recent development of an in vitro lymphocyte colony assay makes it possible to examine variations in the radiosensitivity of humans using peripheral blood lymphocytes (PBL) instead of the skin fibroblast assay. Our recent study (M. Hakoda et al., Mutat. Res. 197, 161-169, 1988) showed that most of the colonies consisted of lymphocytes bearing CD4 or CD8 antigens. Since the fraction of CD4+ and CD8+ cells in PBL differs among individuals, we suspected that individual radiosensitivity might be biased by the different subset frequencies if the dose-survival curves of the CD4+ and CD8+ cells were different from each other. In the present study, CD4+ (helper/inducer T) and CD8+ (suppressor/cytotoxic T) lymphocytes were isolated from PBL and their dose-survival curves were determined. The results showed that the D10 (dose required to reduce the surviving fraction to 10%) was similar for these two types of cells [3.13 +/- 0.10 Gy (mean +/- SD) for CD4+, 3.34 +/- 0.50 Gy for CD8+ and 3.14 +/- 0.17 Gy for the unsorted cells], supporting the use of the whole PBL population for the screening of individuals with altered radiosensitivity.     

Transgenerational genomic instability in the first generation offspring of mice exposed to low-intensity red and near-infrared irradiation in vivo

Transgenerational genomic instability in the first generation offspring of mice exposed to lowintensity infrared laser (632.8 nm) and light-emitting-diode infrared irradiation (850 nm) was investigated in vivo. It was found that the level of spontaneous damage in bone marrow according to the micronucleus test, the level of reactive oxygen species in whole blood, and the mass index of lymphoid organs in all of the descendants of irradiated mice did not increase. After additional X-ray exposure of the progeny at a dose rate of 1.5 Gy, a decrease in the level of damage and the absence of an adaptive response were revealed upon testing according to “radiosensitivity” and the radiation-induced adaptive-response scheme (0.1 + 1.5 Gy), respectively, compared to the descendants of nonirradiated mice. The rate of tumor growth in the offspring of irradiated mice did not differ from that in the descendants of nonirradiated mice, although inhibition of the tumor growth rate was observed in their irradiated parents. The survival rate after irradiation at a dose rate of 6.5 Gy did not differ from both the parents and the control.

     

Radiosensitivity of chromosomes in lymphocytes from Down's syndrome patients

A study was made of the yield of chromosome aberrations in gamma-irradiated G0 peripheral blood lymphocytes from 6 patients with different forms of Down's syndrome. The doses used were from 0.25 to 3.0 Gy. Seven healthy donors of different age made the control group. There was a significant increase in the yield of chromosome exchanges in lymphocytes from all the patients as compared to control. The spontaneous level of chromosome aberrations and the frequency of radiation-induced fragments did not differ from the control values. The yield of exchanges in diploid and trisomic cells from patients with the mosaic form of Down's syndrome did not change significantly as the time of cultivation was raised. The origin of DNA repair defects leading to the increased chromosome radiosensitivity in Down's syndrome is discussed.     

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.     

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.     

The problem of the radiosensitivity of bone marrow cellular elements and assessments of the postradiation kinetics of myelopoiesis (an analysis based on data on the sequelae of the accident at the Chernobyl Atomic Electric Power Station]

On the basis of the data obtained from 61 cases of accidental exposure (0.1-12.5 Gy) at the Chernobyl A.P.S. the kinetics of acute radiation bone-marrow syndrome was analyzed and radiosensitivity of the entire spectrum of human granulocytic compartment cells was estimated. The radiosensitivity estimates were made by a "functional" criterion, developed by the authors, which was based on the comparative ability of irradiated and nonirradiated bone marrow cells of different maturity to produce peripheral blood neutrophils. Changes were found in physiology of myeloid cells during their maturation: the maturation mechanism, for cells of the committed pool, was "attached" to the division process, whereas these processes were independent for cells of the dividing and maturing pool. It is once again confirmed that the transit time of a maturing myeloid cell, to begin with the primarily committed one and to end with a peripheral blood neutrophil, is not constant and lasts normally for 32 days.     

Initial slope of radiation survival curves is characteristic of the origin of primary and established cultures of human tumor cells and fibroblasts

The published survival curves of 110 human tumor cell lines and 147 nontransformed human fibroblast strains have been reanalyzed using three different statistical methods: the single hit multitarget model, the linear-quadratic model, and the mean inactivation dose. The 110 tumor cell lines were classified in two ways: (a) into three categories defined by clinical radiocurability criteria, and (b) into seven categories based on histopathology. The 147 fibroblast strains were divided into eight genetic groups. Differences in the radiosensitivities of both the tumor cell and fibroblast groups could be demonstrated only by parameters that describe the slopes of the initial part of the survival curves. The capacity of the survival level to identify significant differences between groups was dose dependent over the range 1 to 6 Gy. This relationship showed a bell-shaped curve with a maximum at 1.5 Gy for the tumor cell lines and 3 Gy for the fibroblasts. Values for intrinsic radiosensitivity for a number of groups of tumors have also been obtained by primary culture of tumor cells. These values are strictly comparable to those obtained by clonogenic methods. This confirms that intrinsic radiosensitivity is a determinant of the response of tumor cells to radiotherapy and suggests that tissue culture methods may be used as a predictive assay.     

The combined effects of dietary supplements and low-dose-rate high-LET radiation on mice in vivo

The purpose of this study was to investigate the combined actions of food supplements and lowdose-rate high-LET radiation on radiosensitivity, induction of the adaptive response, and tumor growth in SHK mice in vivo. The animals were irradiated with 0.11 Gy (0.005 Gy/day) of low-dose-rate high-LET radiation behind the concrete shield of a 70 GeV proton accelerator (Protvino, Moscow oblast). Four groups of the mice were fed with selected products (soy meat, buckwheat, lettuce leaves, and a drug based on cod-liver oil) during the entire irradiation period (22 days). The results of the study indicate that the mice with diets containing soy meat, buckwheat, and lettuce leaves in contrast to those fed with a diet containing cod-liver oil had reduced sensitivity to X-radiation at a dose rate of 1.5 Gy and a significant slowdown in the growth of the Ehrlich carcinoma. The combined effect of high-LET radiation and the food supplements mentioned above (except for the cod-liver oil) reduced the sensitivity of the mice to the irradiation at a dose rate of 1.5 Gy, induced the adaptive response, and caused a decrease in the growth rate of the Ehrlich carcinoma in contrast to the mice that were only irradiated with high-LET radiation.