Induction of micronuclei and anaphase aberrations by cytochalasin B in human lymphocyte cultures

The frequency of micronucleated cells in isolated 72-h human lymphocyte cultures treated with cytochalasin B (Cyt-B; 1.5-6 micrograms/ml for the last 28 h) was 9-21 times higher (mean 14.6 times) among multinucleate than binucleate cells. At 3 micrograms/ml, the concentration of Cyt-B originally recommended for the human lymphocyte micronucleus assay, the frequency of micronucleated multinucleate cells was 8.5%, while 0.7% of the binucleate cells had a micronucleus. Although no dose-dependent induction of micronuclei could be observed for either of the cell types, increase in the concentration of Cyt-B was associated with a decrease in the ratio of multinucleate to binucleate cells. Treatment with Cyt-B (1.5-12 micrograms/ml) increased the frequency of anaphase cells with aberrations, especially lagging chromatids. This finding was explained by a dose-dependent increase in multipolar (greater than or equal to 3 poles) divisions which had a high frequency of anaphase aberrations (39-53%), irrespective of the concentration of Cyt-B. Bipolar anaphases did not show a significant increase in aberrant cells, although a suggestive dependence on the concentration of Cyt-B was observed. The findings indicate that the high frequency of micronuclei in multinucleate lymphocytes produced by Cyt-B is due to mitotic errors arising when bi- (and multi-) nuclear cells divide. To avoid possible artifactually high micronucleus frequencies due to inclusion of cells that have divided greater than or equal to 2 times in the presence of Cyt-B, it is recommended that, in the human lymphocyte micronucleus assay using the cytokinesis-block method, the cell culture time is reduced to minimize the frequency of such cells and that only good preparations and regularly shaped binucleates are included in the analysis.     

预测鼻咽癌放射敏感性的CB微核法

本文介绍一种预测鼻咽癌放射敏感性的方法——CB微核法。以该法评介了鼻咽癌细胞株及20例鼻咽癌患者活检标本的放射敏感性,随着剂量增加,微核率也明显增加。CB微核的剂量-效应与克隆法的剂量效应呈高度负相关(r=-0.970)。活检标本微核实验表明,20例中8例反应较高,7例中等,5例反应偏 低;随访表明,反应偏低者中已有2例有复发倾向。由于CB微核法具有简便、快速、正确性较高等优点,适于临床应用。     

The in vitro micronucleus technique

The study of DNA damage at the chromosome level is an essential part of genetic toxicology because chromosomal mutation is an important event in carcinogenesis. The micronucleus assays have emerged as one of the preferred methods for assessing chromosome damage because they enable both chromosome loss and chromosome breakage to be measured reliably. Because micronuclei can only be expressed in cells that complete nuclear division a special method was developed that identifies such cells by their binucleate appearance when blocked from performing cytokinesis by cytochalasin-B (Cyt-B), a microfilament-assembly inhibitor. The cytokinesis-block micronucleus (CBMN) assay allows better precision because the data obtained are not confounded by altered cell division kinetics caused by cytotoxicity of agents tested or sub-optimal cell culture conditions. The method is now applied to various cell types for population monitoring of genetic damage, screening of chemicals for genotoxic potential and for specific purposes such as the prediction of the radiosensitivity of tumours and the inter-individual variation in radiosensitivity. In its current basic form the CBMN assay can provide, using simple morphological criteria, the following measures of genotoxicity and cytotoxicity: chromosome breakage, chromosome loss, chromosome rearrangement (nucleoplasmic bridges), cell division inhibition, necrosis and apoptosis. The cytosine-arabinoside modification of the CBMN assay allows for measurement of excision repairable lesions. The use of molecular probes enables chromosome loss to be distinguished from chromosome breakage and importantly non-disjunction in non-micronucleated binucleated cells can be efficiently measured. The in vitro CBMN technique, therefore, provides multiple and complementary measures of genotoxicity and cytotoxicity which can be achieved with relative ease within one system. The basic principles and methods (including detailed scoring criteria for all the genotoxicity and cytotoxicity end-points) of the CBMN assay are described and areas for future development identified.     

Measurement of micronuclei in lymphocytes

The micronucleus technique has been proposed as a method for measurement of chromosomal damage in mitogen-stimulated human lymphocytes. Micronuclei require one cell division to be expressed and, consequently, the conventional micronucleus technique is very imprecise since the cells which have undergone only one division, and the micronuclei in them, cannot be identified separately from the total population of lymphocytes. To overcome this problem, two methods were developed to identify cells which have undergone their first mitosis. Using an autoradiographic technique, lymphocytes were pulse-labelled with [3H]thymidine at 48 h of culture, allowed to proceed through mitosis, identified by autoradiography between 72 and 84 h and micronuclei were scored in them. It was not possible to select a concentration of radiolabel which did not itself produce micronuclei and consequently the method was of no value for measuring pre-existing chromosomal damage present in vivo. However, it was capable of quantitating micronuclei produced by irradiation of lymphocytes in vitro. In the second method, cytokinesis was blocked using cytochalasin B. Micronuclei were scored in cytokinesis-blocked cells. These were easily recognisable owing to their binucleate appearance and a large number could be accumulated by adding 3.0 micrograms/ml cytochalasin B at 44 h and scoring at 72 h. Cytochalasin B did not itself produce micronuclei. The cytokinesis-block method was simple to perform; the 'in vivo' micronucleus frequency in normal individuals was 4.4 +/- 2.6 micronuclei/500 cytokinesis-blocked cells; and for lymphocytes irradiated in vitro there was a linear relationship between dose of radiation and number of induced micronuclei. The cytokinesis-block method appears to be the procedure of choice for quantitating micronuclei in lymphocytes.     

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.     

Binucleate cell formation correlates to loss of colony-forming ability in X-irradiated cultured mammalian cells

The relationship between binucleate cell formation and the loss of colony-forming ability was examined in several cultured mammalian cell lines irradiated with X rays. The maximum fraction of binucleate cells after X irradiation increased dose-dependently within the range in which reproductive cell death might predominate over interphase cell death. When the logarithm of percentage survival was plotted against the percentage binucleate cells, a similar correlation was found for all cell lines tested, with the exception of mouse leukemia L5178Y cells, the most radiosensitive cells used. These observations suggest that the fraction of binucleate cells in the cell population can serve as a measure of cellular radiation damage.     

The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells

The assessment of tumor radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. Therefore, the degree of correlation between radiation-induced DNA damage, as measured by the alkaline and the neutral comet assays, and the clonogenic survival of different human tumor cells was studied. Further, tumor radiosensitivity was compared with the expression of genes associated with the cellular response to radiation damage. Five different human tumor cell lines were chosen and the radiosensitivity of these cells was established by clonogenic assay. Alkaline and neutral comet assays were performed in γ-irradiated cells (2-8Gy; either acute or fractionated). Quantitative PCR was performed to evaluate the expression of DNA damage response genes in control and irradiated cells. The relative radiosensitivity of the cell lines assessed by the extent of DNA damage (neutral comet assay) immediately after irradiation (4Gy or 6Gy) was in agreement with radiosensitivity pattern obtained by the clonogenic assay. The survival fraction of irradiated cells showed a better correlation with the magnitude of DNA damage measured by the neutral comet assay (r=-0.9; P<0.05; 6Gy) than evaluated by alkaline comet assay (r=-0.73; P<0.05; 6Gy). Further, a significant correlation between the clonogenic survival and DNA damage was observed in cells exposed to fractionated doses of radiation. Of 15 genes investigated in the gene expression study, HSP70, KU80 and RAD51 all showed significant positive correlations (r=0.9; P<0.05) with tumor radiosensitivity. Our study clearly demonstrated that the neutral comet assay was better than alkaline comet assay for assessment of radiosensitivities of tumor cells after acute or fractionated doses of irradiation.     

A method for the selective measurement of the radiosensitivity of quiescent cells in solid tumors--combination of immunofluorescence staining to BrdU and micronucleus assay.

C3H/He mice bearing the SCC VII tumor were irradiated after being given 10 injections of 5-bromo-2'-deoxyuridine (BrdU) to label all proliferating cells in the tumors, and the tumors were then excised and trypsinized. The tumor cell suspensions were incubated with cytochalasin-B (which blocks cytokinesis), and the micronucleus frequency in unlabeled cells was determined using immunofluorescence staining to BrdU. The micronucleus frequency was then used to calculate the surviving fraction of the unlabeled cells, using the regression line relating the micronucleus frequency to the surviving fraction determined separately for the total tumor cell population. Using this technique, a cell survival curve could be determined for the unlabeled cells, which were regarded as the quiescent cells. Assays performed both immediately after and 24 h after irradiation of normally-aerated tumors showed that unlabeled cells were more radioresistant and had a greater capacity for repair of potentially lethal damage than the tumor cell population as a whole. Moreover, when the assay was performed immediately after the irradiation of both normally-aerated and hypoxic tumors, it was found that unlabeled cells had a much higher hypoxic fraction than the tumor cell population as a whole. This appears to be a useful method for determining the responses of quiescent cells in solid tumors to various treatments.     

Cytokinesis-block micronucleus method in human lymphocytes: effect of in vivo ageing and low dose X-irradiation

The cytokinesis-block micronucleus technique was developed to overcome the kinetic problems inherent in the use of human lymphocytes for micronucleus assays. Using this technique the number of spontaneous micronuclei in lymphocytes from 42 individuals aged between 20 and 85 years was studied and was found to increase at a rate of 4.3% per year. Comparison with the results obtained with the conventional micronucleus assay confirmed that the conventional method markedly underestimates this age effect. The sensitivity of the cytokinesis-block method was determined by studying the effect of low-dose (less than 50 rad) X-irradiation. The results indicated that the dose-response was linear and a single in vitro exposure to 5 rad of X-rays could be unequivocally detected. We concluded that the cytokinesis-block micronucleus method is more sensitive and precise than the conventional micronucleus method and classical metaphase analysis, and that it will be of value for detecting chromosome damage induced in vivo by genotoxic agents.     

Response of lymphosarcoma LS/BL cells to continuous irradiation

Mouse lymphosarcoma LS/BL cells growing as an ascites tumor in the peritoneal cavity of C57BL mice were continuously irradiated in vivo at a low exposure rate of 1.2 Gy per day (5 rad/hr). The growth of the ascites tumor evaluated by direct counting of the cells in the peritoneal cavity and their capacity to form colonies in livers declined with increasing time of continuous irradiation. The radiosensitivity and repair ability of LS/BL cells were studied by a serial dilution method using host survival time as the end point and by the liver colony assay. The radiosensitivity of continuously irradiated LS/BL-CI cells showed no remarkable change as measured by the Do values, but from the 150th week of irradiation the initial shoulder on the survival curves appeared and its width increased with time of exposure. The extrapolation number (n) increased from 1.0 to 8.4 after 350 weeks of irradiation. The reappearance of the initial shoulder was proved with the split-dose technique.     

Farnesyltransferase inhibitors potentiate the antitumor effect of radiation on a human tumor xenograft expressing activated HRAS

Successful radiosensitization requires that tumor cells become more radiosensitive without causing an equivalent reduction in the survival of cells of the surrounding normal tissues. Since tumor cell radiosensitivity can be influenced by RAS oncogene activation, we have hypothesized that inhibition of oncogenic RAS activity would lead to radiosensitization of tumors with activated RAS. We previously showed in tissue culture that prenyltransferase treatment of cells with activated RAS resulted in radiosensitization, whereas treatment of cells with wild-type RAS had no effect on radiation survival. Here we ask whether the findings obtained in vitro have applicability in vivo. We found that treatment of nude mice bearing T24 tumor cell xenografts with farnesyltransferase inhibitors resulted in a significant and synergistic reduction in tumor cell survival after irradiation. The regrowth of T24 tumors expressing activated RAS was also significantly prolonged by the addition of treatment with farnesyltransferase inhibitors compared to the regrowth after irradiation alone. In contrast, there was no effect on the radiosensitivity of HT-29 tumors expressing wild-type RAS. These results demonstrate that specific radiosensitization of tumors expressing activated RAS oncogenes can be obtained in vivo.     

Response of human squamous cell carcinoma xenografts of different sizes to irradiation: relationship of clonogenic cells, cellular radiation sensitivity in vivo, and tumor rescuing units

The relationship of clonogenic cells, cellular radiation sensitivity at tumor control does in vivo, and tumor rescuing units at different tumor sizes was investigated in the human squamous cell carcinoma FaDu growing in NCr/Sed nude mice. The composition of the tumors was determined in single cell suspensions and compared to tumor control data after single-dose irradiation. To avoid the influence of varying oxygen concentrations in the tumors, all irradiations were performed under clamp hypoxia. Nude mice and animals further immunosuppressed by 6-Gy whole-body irradiation were used to assess the immunological effects. The numbers of total cells, cells excluding trypan blue, host cells, and colony-forming cells increased linearly with the weight of FaDu tumors. Comparable results were obtained for cell suspensions prepared from tumors growing in nude of pretreated nude mice. The radiation dose required to control 50% of tumors (TCD50) of different sizes between 36 and 470 mm3 increased from 52.1 to 60.1 Gy when the tumors were maintained in normal nude mice and from 50.8 to 61.3 Gy in whole-body-irradiated mice. The D0 of FaDu cells in vivo was calculated by regression analysis of TCD50 vs the logarithm of the clonogenic cell number, assuming an oxygen enhancement ratio of 3.0. The resultant D0S of 1.1 and 1.2 Gy in vivo correspond well to the radiosensitivity of FaDu cells in vitro determined previously. Assuming the single-hit multitarget model of cell killing and extrapolation numbers between 2 and 20, the mean number of tumor rescuing units would be 10(5) to 10(6) for a 100-mm3 tumor growing in whole-body-irradiated nude mice. Comparison of the number of tumor rescuing units to the estimated number of clonogenic cells does not conflict with the assumption that every surviving clonogenic cell is able to repopulate FaDu tumors after irradiation; however, it seems more likely that more than one clonogenic cells is necessary. The proportion of tumor rescuing units in the clonogenic cell population is independent of tumor size.     

Development of a method for assessing micronucleus induction in a 3D human skin model (EpiDerm)

To meet the requirements of the EU 7th Amendment to the Cosmetics Directive, manufacturers of cosmetics products will need to ascertain the safety of ingredients using non-animal methods. Starting in 2009, in vivo genotoxicity tests for cosmetics ingredients will not be allowed. Skin is a target area of interest for many cosmetic products because of its relatively high exposure. Therefore, it would be beneficial to have a non-animal, skin-based genotoxicity assay, especially one that utilized human skin in vitro. In this paper, we describe the development of a reproducible micronucleus assay that uses EpiDerm engineered human skin constructs (MatTek Corp., Ashland, MA). We describe methods for isolating single cells from the 3D skin model and for processing the cells for microscopic analysis of micronuclei (MN). In addition, since little was known about the kinetics of the dividing keratinocytes in the EpiDerm model, we evaluated whether cytochalasin B (Cyt-B) could be used to distinguish the population of dividing cells allowing the development of a micronucleus assay in binucleated cells. We found that the frequency of binucleated cells increased both with time and with increasing concentration of Cyt-B. After a 48-h exposure, 30-50% binucleated cells were reproducibly obtained. Finally, we evaluated micronucleus induction using the model genotoxicants mitomycin C (MMC) and vinblastine sulfate (VB). The background frequency of MN is very low and reproducible in this model, and statistically significant increases in the frequency of micronucleated cells were induced by both MMC and VB. These are initial steps in developing a routine "in vivo-like" assay for chromosomal damage in human tissue. It is hoped that other investigators utilize these methods to further the understanding of this potentially valuable new non-animal method.     

Use of the cytokinesis-block micronucleus method in mouse splenocytes

Mouse splenocytes have been used in the cytokinesis-block method for the evaluation of micronuclei induced by mutagenic agents in vitro as well as in vivo. Stimulation with concanavalin A for 48 h followed by 16-24-h treatment with 5 micrograms/ml cytochalasin B was found to be an optimum condition to obtain micronuclei in the binucleated splenocytes after the cells were cultured in vitro. Under the above conditions splenocytes from mice pretreated with a single i.p. injection of cyclophosphamide gave a significant increase in micronucleus production. This increase was dependent on the dose of cyclophosphamide (r = 0.99). A dose of 50 mg/kg resulted in 22% of the binucleated cells producing micronuclei, more than 20 times the level in the untreated control. The increase was also dependent on the time of cyclophosphamide injection before removal of the spleen. A duration of 4-8 h after cyclophosphamide injection gave rather sharp optimum values for the production of micronuclei. When splenocytes from non-treated mice were treated with mitomycin C together with cytochalasin B in the above in vitro condition, there was a significant increase in micronucleus production in the binucleated cells. It was also dependent on the dose of mitomycin C (r = 0.975) and a dose of 0.5 micrograms/ml resulted in a more than 20-fold increase over the untreated control. Thus, the use of mouse splenocytes in the cytokinesis-block micronucleus assay was shown to be sensitive enough for testing mutagenic agents in vivo as well as in vitro.     

Chromosomal aberrations and DNA repair ability of in vitro irradiated white blood cells of monkeys previously exposed to total body irradiation

Six monkeys (Macaca fascicularis) were total-body-irradiated with 60Co (fractionated irradiation of 8 or 10 Gy). Blood samples were collected at different times post total-body irradiation, then in vitro irradiated in order to test whether a prior in vivo irradiation could affect the radiosensitivity of their leukocytes. We suggested in a preliminary report that the enhanced chromosomal radiosensitivity of in vivo irradiated monkeys could be correlated with a DNA repair deficiency (Guedeney et al., 1986). Chromosomal aberrations, the rate of initial strand breaks and their rejoining estimated using a fluorescent assay for DNA unwinding were chosen as the endpoints in this more extensive study. We observed that the yield of dicentrics induced by a subsequent in vitro irradiation was lower than that scored in unirradiated monkeys in few cases (6/22) whereas the number of acentrics was found to be modified in 16 of the 22 samples. An altered DNA repair ability was observed in most but not all blood samples tested. Thus, in view of such intra-individual variability, the results of this more extensive study lead us to conclude that a previous total-body irradiation does not alter the gamma-induced chromosome aberrations and DNA repair ability in a reproducible manner.     

Radiation-induced micronucleus frequency in peripheral blood lymphocytes is correlated with normal tissue damage in patients with cervical carcinoma undergoing radiotherapy

In an effort to find a test to predict the response of normal tissue to radiotherapy, the lymphocyte micronucleus assay was used on blood samples from patients with cervical carcinoma. Peripheral blood samples from 55 patients with advanced-stage (II B-IV B) cervical carcinoma were obtained before radiotherapy. The patients were treated with external-beam radiotherapy followed by high-dose-rate brachytherapy. Acute and late normal tissue reactions were scored and correlated with the micronucleus frequency in lymphocytes after irradiation with 4 Gy in vitro. Great interindividual variability was observed in the radiation-induced lymphocyte micronucleus frequency, especially at 4 Gy. The mean number of micronuclei per 100 binucleated cells in cells irradiated with 4 Gy in vitro was significantly higher in samples from patients who suffered from acute and/or late normal tissue reactions than in those from patients with no reactions (51.0 +/- 17.7 and 29.6 +/- 10.1, respectively). A significant correlation was also found between the micronucleus frequency at 4 Gy and the severity of acute reactions and late reactions. However, the overlap between the micronucleus frequencies of patients with high-grade late normal tissue reactions and low-grade reactions is too great to recommend the micronucleus assay in its present form for routine clinical application.     

Development of an in vitro colony formation assay for the evaluation of in vivo chemotherapy of a rat brain tumor.

An in vitro colony formation assay for the evaluation of in vivo brain tumor therapy has been developed. When plated, disaggregated cells derived from solid tumors proliferated to form relatively homogeneous colonies after a latency period of 2 to 6 days. Increasing concentrations of fetal calf serum enhanced colony-forming efficiency (CFE) with a plateau between 7 and 16%. Supplementation with either irradiated feeder cells (10(3) to 10(5) cells per dish), or medium conditioned by 1 to 3 days of in vitro incubation with the same cell line, doubled the CFE. The density of tumor cells (untreated or previously treated with chemotherapeutic agents) did not affect the CFE when a minimum of 10(4) total cells (tumor plus feeder) were plated. Therefore, in this system the optimal experimental conditions for evaluating chemotherapy and radiotherapy require incubation of disaggregated tumor cells for 12 days in medium containing 10% of fetal calf serum and enough feeder cells to provide a minimum of 10(4) cells per dish. The CFE for untreated tumors was 18 +/- 10% (+/-S.D.), demonstrating that there is significant biological variation. The assay appeared sensitive, with reproducible results, when applied to individual chemically treated tumors. An estimate of the percentage of clonogenic cells affected by in vivo chemotherapy may be obtained by comparing the CFE of cells from treated and untreated tumors. This assay can measure up to a 5 log(10) cell kill, and it should prove to be valuable in developing more effective regimens for the treatment of solid tumors in animals and man.     

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.

     

The relationship between the in vitro chemosensitivity of tumor cells and tumor response in vivo in an experimental tumor model

Cell lines derived from a panel of five histologically distinct murine adenocarcinomas of the colon (MAC) were used to assess whether or not a colony-forming assay could have retrospectively predicted the wide range of in vivo responses to chlorambucil (CHL). The predictive value of the clonogenic assay was significantly improved when fractions (one-tenth) of the plasma drug AUC (from the area under the drug clearance curves); were used to determine clonogenic cell kill in vitro, instead of one-tenth peak plasma drug concentration and total plasma drug AUC exposures. Despite the good correlation between in vitro and in vivo responses observed, the clonogenic assay could not forecast the site-dependent response of MAC 15A to CHL. These site-dependent responses cannot be explained in terms of the inherent sensitivity of tumor cells themselves, suggesting that caution must be applied in the interpretation of in vitro chemosensitivity assays.     

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.