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.     

A dose-effect relationship study for the frequency of binuclear cells with micronuclei in a human lymphocyte culture after gamma irradiation

The method of the cytokinetic blocking was used to determine, in 18 healthy male donors aged 21-35 years, the frequency of binuclear cells with micronuclei that amounted to 1.1 +/- 0.2% (0.3-2.4%) on the average. The dependence of this parameter upon radiation dose (60Co-gamma radiation, 0.05 to 2 Gy) was a linear quadratic function and was described by the following regression equation: Yx = 0.76 + 5.76 D +/- 2.61 D2. With a dose of 0.05 Gy the frequency of binuclear cells with micronuclei exceeded significantly the control level.     

RBE of 10 kV X rays determined for the human mammary epithelial cell line MCF-12A

The dependence of relative biological effectiveness (RBE) on photon energy is a topic of extensive discussions. The increasing amount of in vitro data in the low-energy region indicates this to be a complex dependence that is influenced by the end point and cell line studied. In the present investigation, the RBE of 10 kV X rays (W anode) was determined relative to 200 kV X rays (W anode, 0.5 mm copper filter) for cell survival in the dose range 1-10 Gy and for induction of micronuclei in the range 0.5-3.6 Gy for MCF-12A human mammary epithelial cells. The RBE for cell survival was found to increase with decreasing dose, being 1.21+/-0.03 at 10% survival. Considerably higher values were obtained for micronucleus induction, where the RBE(M) obtained from the ratio of the linear coefficients of the dose-effect curves was 2.6+/-0.4 for the fraction of binucleated cells with micronuclei and 4.1+/-1.0 for the number of micronuclei per binucleated cell. These values, together with our previous data, support a monotonic increase in RBE with decreasing photon energy down to the mean energy of 7.3 keV used in the present study.     

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

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

Genome instability in the F1-progeny of mice irradiated by ionizing radiation as determined by micronucleus assay

The F1-progeny of BALB/c male mice chronically exposed to low-dose gamma-radiation (0.1; 0.25 and 0.5 Gy; dose rate 0.01 Gy/day) as well as the F1-progeny of females exposed to acute X-radiation (0.5; 1.0 and 2.0 Gy; dose rate 0.1 Gy/min) shown the significant elevated micronuclei frequencies in bone marrow erythrocytes, as compared to the F1-progeny of unirradiated males and females. The increase in the micronuclei frequency in the F1-progeny was determined by the dose of irradiation of parents. The values of elevated micronuclei frequency in the F1-progeny of chronically irradiated males and acutely irradiated females for a dose of 0.5 Gy were comparable. The micronuclei frequencies in the F1-progeny of irradiated females and males for this dose were in 1.5 and in 1.6 times higher than ones in the F1-progeny of unirradiated mice correspondingly. The results suggest the possibility of transfer of genome instability from irradiated parents to the somatic cells of the F1-progeny via non-lethally damaged germ cells of parents.     

Micronucleus induction in mammalian cell cultures treated with ionizing radiations

Summary Exponentially growing and plateau phase cultures of Ehrlich ascites tumor cells (suspension strain) were treated with either fast electrons, X-rays, fast neutrons or Am-241-alpha-particles in a dose range from about 0.02 Gy to 1 Gy and for comparison also at higher doses. After the first post-irradiation division, cells were scored for the presence of micronuclei and the micronucleus fraction as well as the number of micronuclei/cell was determined. Micronuclei were counted using the DNA specific stain H 33258 in a fluorescence microscope. A comparison with cytofluorometric measurements established that microscopic detection accounted for up to 90% of all micronuclei present within a sample, the rest probably being hidden in direct observation by the main nucleus.Dose response curves based on the micronucleus fraction as well as on the number of micronuclei/cell were found to be linear in the whole dose range tested at low and at high ionization density. Linearity was maintained also when repair of primary lesions was promoted or suppressed. The RBE of alpha-particles compared with X-rays was dependent on the time of fixation and was at a maximum immediately after the first division (RBE = 4.8 ± 0.5). Micronucleus distribution showed overdispersion relative to Poissonian statistics with every radiation quality used, in accordance with earlier observations on the distribution of acentric fragments in irradiated cultures.     

The effect of 835.62 MHz FDMA or 847.74 MHz CDMA modulated radiofrequency radiation on the induction of micronuclei in C3H 10T(1/2) cells

To determine if radiofrequency (RF) radiation induces the formation of micronuclei, C3H 10T(1/2) cells were exposed to 835.62 MHz frequency division multiple access (FDMA) or 847.74 MHz code division multiple access (CDMA) modulated RF radiation. After the exposure to RF radiation, the micronucleus assay was performed by the cytokinesis block method using cytochalasin B treatment. The micronuclei appearing after mitosis were scored in binucleated cells using acridine orange staining. The frequency of micronuclei was scored both as the percentage of binucleated cells with micronuclei and as the number of micronuclei per 100 binucleated cells. Treatment of cells with cytochalasin B at a concentration of 2 microg/ml for 22 h was found to yield the maximum number of binucleated cells in C3H 10T(1/2) cells. The method used for the micronucleus assay in the present study detected a highly significant dose response for both indices of micronucleus production in the dose range of 0.1-1.2 Gy and it was sensitive enough to detect a significant (P > 0.05) increase in micronuclei after doses of 0.3 Gy in exponentially growing cells and after 0.9 Gy in plateau-phase cells. Exponentially growing cells or plateau-phase cells were exposed to CDMA (3.2 or 4.8 W/kg) or FDMA (3.2 or 5.1 W/kg) RF radiation for 3, 8, 16 or 24 h. In three repeat experiments, no exposure condition was found by analysis of variance to result in a significant increase relative to sham-exposed cells either in the percentage of binucleated cells with micronuclei or in the number of micronuclei per 100 binucleated cells. In this study, data from cells exposed to different RF signals at two SARs were compared to a common sham-exposed sample. We used the Dunnett's test, which is specifically designed for this purpose, and found no significant exposure-related differences for either plateau-phase cells or exponentially growing cells. Thus the results of this study are not consistent with the possibility that these RF radiations induce micronuclei.     

The human lymphocyte micronucleus assay. Response of cord blood lymphocytes to gamma-irradiation and bleomycin

The induction of micronuclei in human cord blood lymphocytes by treatment with gamma-irradiation and bleomycin has been measured. Culture durations which gave peak MN frequencies were determined. The lowest tested doses, 0.1 Gy irradiation and 1.25 micrograms/ml bleomycin, produced significant increases in the frequency of micronuclei. The spontaneous frequency of micronucleated lymphocytes in 28 cord blood samples ranged between 0.5 and 9.5 per thousand lymphocytes, with a modal value of 2.5. The method is evaluated for its potential usefulness in monitoring populations for chromosome breakage.     

RPA foci are associated with cell death after irradiation

MacPhail, S. H. and Olive, P. L. RPA Foci are Associated with Cell Death after Irradiation. Radiat. Res. 155, 672-679 (2001). Complexes containing replication protein A (RPA) were observed in human TK6 and WIL-2NS lymphoblast cells and SiHa cervical carcinoma cells exposed to 250 kV X rays. Image analysis of individual cells with fluorescence-tagged anti-RPA antibodies was used to measure numbers of discrete foci per cell. RPA foci formed in S-phase cells in response to radiation doses as low as 0.5 Gy, and the number of foci/nucleus was linearly related to dose up to 50 Gy. The maximum number of cells with foci occurred 4-8 h after exposure to 4 Gy, and subsequently declined. However, the number of RPA foci per nucleus (in those cells with foci) reached a maximum after 2-4 h. Apoptotic nuclei from irradiated TK6 and WIL-2NS cells initially contained foci, but these were lost as degradation continued. Radiation-induced micronuclei in SiHa cells were greatly enriched for RPA foci, and cells with nuclei without foci often contained micronuclei with multiple RPA foci. In SiHa cells examined up to 7 days after 4 Gy, RPA foci reappeared in one or more cells in up to 90% of the surviving colonies, and some cells contained 150 or more distinct foci. Reappearance of these complexes could be indicative of radiation-induced genomic instability. These results are consistent with the idea that RPA foci observed several hours after irradiation represent irreparable lesions and as such might be useful in identifying radiosensitive cells.     

Establishment of dose-response relationships between doses of Cs-137 gamma-rays and frequencies of micronuclei in human peripheral blood lymphocytes

It has been suggested that the yield of micronuclei in human peripheral blood lymphocytes could be used as a biological dosimeter in cases of radiation exposure. In the present study micronuclei were induced in lymphocytes by exposing human blood samples in vitro to various doses of Cs-137 gamma-rays. The blood samples were then cultivated using the cytokinesis block method. Coded programs were employed to establish the relationships between the frequencies of micronuclei and various doses of gamma-rays. The best fit was obtained by the linear-quadratic model, Y = c + aD + bd2, where Y is the yield of micronuclei, D is the dose in Gy and c, a, b, are constants. It seems there is a correlation between the yields of MN in mononuclear cells and the corresponding doses of radiation. Therefore an attempt was made to include these MN in the calculation of the dose-response relationship.     

The human lymphocyte micronucleus assay: Response of cord blood lymphocytes to γ-irradiation and bleomycin

The induction of micronuclei in human cord blood lymphocytes by treatment with γ-irradiation and bleomycin has been measured. Culture durations which gave peak MN frequencies were determined. The lowest tested doses, 0.1 Gy irradiation and 1.25 μg/ml bleomycin, produced significant increases in the frequency of micronuclei. The spontaneous frequency of micronucleated lymphocytes in 28 cord blood samples ranged between 0.5 and 9.5 per thousand lymphocytes, with a modal value of 2.5. The method is evaluated for its potential usefulness in monitoring populations for chromosome breakage.     

Oncogenic transformation by fractionated doses of neutrons

Oncogenic transformation was assayed after C3H 10T1/2 cells were irradiated with monoenergetic neutrons; cells were exposed to 0.23-, 0.35-, 0.45-, 5.9-, and 13.7-MeV neutrons given singly or in five equal fractions over 8 h. At the biologically effective neutron energy of 0.45 MeV, enhancement of transformation was evident with some small fractionated doses (below 1 Gy). When transformation was examined as a function of neutron energy at 0.5 Gy, enhancement was seen for cells exposed to three of the five energies (0.35, 0.45, and 5.9 MeV). Enhancement was greatest for cells irradiated with 5.9-MeV neutrons. Of the neutron energies examined, 5.9-MeV neutrons had the lowest dose-averaged lineal energy and linear energy transfer. This suggests that enhancement of transformation by fractionated low doses of neutrons may be radiation-quality dependent.     

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.     

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).     

Characteristic cytogenetic effects of small doses of ionizing radiation

A study was made of the frequency of chromosome aberrations in human lymphocyte culture after gamma-irradiation (60Co) with doses ranging from 0.05 to 1.0 Gy at dose--rates of 0,005, 0.05 and 0.5 Gy/min. The frequency of structural changes in chromosomes at low doses was higher than it was expected in the case of extrapolating the effect produced by high to low doses of radiation; within the dose range from 0.1 to 0.5 Gy a plateau was registered for aberrations of the exchange type (dicentrics and rings). The abnormal character of the dose dependence of the yield of chromosome aberrations persisted with all three dose - rates under study.     

Vicia faba chromosome damage induced by low doses of gamma radiation

The rate of radiation damage to chromosomes by low doses of gamma rays (0.01-0.30 Gy) was studied in the root tips ofVicia faba. As criteria of the effect of ionizing radiation, the frequency of sister chromatid exchanges (SCEs), incidence of chromosomal aberrations and the number of micronuclei were evaluated and compared in irradiated cells. The results obtained confirmed that the analysis of SCEs did not represent an efficient indicator of radiation damage to chromosomes. On the contrary, the formation of chromosomal aberrations and micronuclei was effectively stimulated by low radiation doses, there being linear dose-effect relationships in the low doses region used.     

Radiation-induced bystander effect in healthy G(o) human lymphocytes: biological and clinical significance

To study the bystander effects, G(0) human peripheral blood lymphocytes were X-irradiated with 0.1, 0.5 and 3 Gy. After 24h, cell-free conditioned media from irradiated cultures were transferred to unexposed lymphocytes. Following 48 h of medium transfer, viability, induction of apoptosis, telomere shortening, reactive oxygen species (ROS) levels and micronuclei (after stimulation) were analyzed. A statistically significant decrement in cell viability, concomitant with the loss of mitochondrial membrane potential, telomere shortening, increases in hydrogen peroxide (H(2)O(2)) and superoxide anion (O(2)(-)) with depletion of intracellular glutathione (GSH) level, and higher frequencies of micronuclei, were observed in bystander lymphocytes incubated with medium from 0.5 and 3 Gy irradiated samples, compared to lymphocytes unexposed. Furthermore, no statistically significant difference between the response to 0.5 and 3 Gy of irradiation in bystander lymphocytes, was found. However, when lymphocytes were irradiated with 0.1 Gy, no bystander effect with regard to viability, apoptosis, telomere length, and micronuclei was observed, although a high production of ROS level persisted. Radiation in the presence of the radical scavenger dimethyl sulfoxide (DMSO) suppressed oxidative stress induced by 3 Gy of X-rays with the effective elimination of bystander effects, suggesting a correlation between ROS and bystander signal formation in irradiated cells. The data propose that bystander effect might be mostly due to the reactions of radiation induced free radicals on DNA, with the existence of a threshold at which the bystander signal is not operative (0.1 Gy dose of X-rays). Our results may have clinical implications for health risk associated with radiation exposure.     

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.     

Adaptive doses of irradiation—an approach to a new therapy concept for bladder cancer?

Radiation adaptive response in terms of induced radioresistance or hyperradiosensitivity, has been studied in HCV29 (human bladder epithelium) and RT4 (human bladder carcinoma) cell lines. After pre-irradiation doses of 0.05 Gy or 0.1 Gy, HCV29 cells showed induced radioresistance, whereas after pre-irradiation doses of 0.05 Gy, 0.1 Gy, 0.2 Gy, and 0.5 Gy, the RT4 cells clearly showed hyperradiosensitivity. On the basis of these results, an approach has been developed that may lead to a concept for a new radiotherapeutic regimen of bladder cancer that includes protection of normal cells, on the one hand, and the potential of tumor cell damage, on the other hand. These findings need to be confirmed in further studies for the benefit of the patients.     

Establishment of dose-response relationships between doses of Cs-137 γ-rays and frequencies of micronuclei in human peripheral blood lymphocytes

It has been suggested that the yield of micronuclei in human peripheral blood lymphocytes could be used as a biological dosimeter in cases of radiation exposure. In the present study micronuclei were induced in lymphocytes by exposing human blood samples in vitro to various doses of Cs-137 γ-rays. The blood samples were then cultivated using the cytokinesis block method. Coded programs were employed to establish the relationships between the frequencies of micronuclei and various doses of γ-rays. The best fit was obtained by the linear-quadratic model, Y = c + aD + bd², where Y is the yield of micronuclei, D is the dose in Gy and c, a, b, are constants. It seems there is a correlation between the yields of MN in mononuclear cells and the corresponding doses of radiation. Therefore an attempt was made to include these MN in the calculation of the dose-response relationship.