Adaptive response in different mitotic cycles after irradiation

The frequency of cells with chromosome aberrations and the number of aberrations per cell have been studied by metaphase analysis in the nonirradiated progeny of irradiated human blood lymphocytes. DNA fragmentation (DNA double-stranded breaks) has been investigated by DNA comet assay. To study the adaptive response (AR), PHA-stimulated lymphocytes were irradiated by the adaptive dose (0.05 Gy) in 24 h and by challenge dose (1 Gy) in 48 h after stimulation. The first through fourth mitoses were identified by 5-bromodeoxyuridine. It was found that the frequency of chromosome aberrations and double-strand breaks were increased in all mitotic cycles after the challenge irradiation. In most individuals, the adaptive response is induced by adaptive and challenge irradiations in the first and the second mitotic cycles (48 and 72 h after stimulation, respectively); however, it is absent in the third and the fourth mitoses. In the first mitosis (1Gy in 48 h after stimulation), only chromatid aberrations are observed; chromosome aberrations were registered in subsequent mitoses. DNA comet assay showed that the adaptive response was obvious at 48–72 h, but not 96 h, after stimulation. It can be concluded that the nonirradiated progeny of irradiated lymphocytes have genomic instability. The adaptive response is manifested up to the third mitosis and is explained by the decreasing number of chromatid and chromosome aberrations and DNA fragmentation. We suppose that double-stranded DNA breaks may be damage signals for the induction of adaptive response.     

Cellular reactions induced by chronic exposure to 14C beta-rays

In V-79 cells the Effects of chronic exposure on induction of chromosome aberrations and abnormal metaphases as well as on efficiency of subsequent exposure to 2 Gy gamma-rays were investigated. It was found that chronic exposure increased the yield of chromosome aberrations as well as abnormal metaphases (spread-metaphases and apoptotic metaphases). In spite of the level of damages in cells, the chronic beta-exposure protected cells against the additional induction of chromosomal aberrations by subsequent exposure to higher acute dose of gamma-irradiation. Cytogenetic adaptive response was retained in the surviving progeny of irradiated cells which were cultured in free medium during 40, 70 days or one year after chronic exposure. At this time the level of residual chromosome aberrations, colony forming ability and distribution of the cells by the number of chromosomes were almost the same as in unirradiated cells. However, the high level of abnormal metaphases and half as much of cells in colony in the surviving progeny of irradiated cells in comparison with unirradiated cell, allow us to suggest that the chronic exposure induced the selection of adaptive forms in condition of the higher level of radiation.     

Chromosome aberrations in relation to radiation dose following partial-body exposures in three populations

Structural chromosome aberrations were evaluated in peripheral blood samples obtained from three populations exposed to partial-body irradiation. These included 143 persons who received radiotherapy for enlarged thymus glands during infancy and 50 sibling controls; 79 persons irradiated for enlarged tonsils and 81 persons surgically treated for the same condition during childhood; and 77 women frequently exposed as young adults to fluoroscopic chest X rays during lung collapse treatment for tuberculosis (TB) and 66 women of similar ages treated for TB with other therapies. Radiation exposures occurred 30 and more years before blood was drawn. Doses to active bone marrow averaged over the entire body were 21, 6, and 14 cGy for the exposed thymic, tonsil, and TB subjects, respectively. Two hundred metaphases were scored for each subject, and the frequencies of symmetrical (stable) and asymmetrical (unstable) chromosome aberrations were quantified in 97,200 metaphases. Cells with stable aberrations were detected with greater frequency in the irradiated subjects compared with nonirradiated subjects in all three populations, and an overall test for an association between stable aberrations and partial-body ionizing radiation was highly significant (P less than 0.001). We found no evidence that radiation-induced aberrations varied by age at exposure. These data show that exposure of children or young adults to partial-body fractionated radiation can result in detectable increased frequencies of stable chromosome aberrations in circulating lymphocytes 30 years later, and that these aberrations appear to be informative as biological markers of population exposure.     

The analysis of chromosome lesions in lymphocytes of Hodgkin's lymphoma patients after chemotherapy and radiation therapy

The analysis of chromosome lesions in peripheral blood lymphocytes of Hodgkin's lymphoma (HL) patients after chemotherapy and chemotherapy with the subsequent course of radiation therapy is carried out. Is shown, that the mean aberration frequency was significantly higher in HL patients after chemotherapy (7.20 +/- 0.58 per 100 metaphases) than in non-treated HL patients (4.80 +/- 0.54, p < 0.01). The subsequent carrying out of radiation therapy enlarges number of chromosome aberrations on 100 metaphases up to 46.7 +/- 10.7 (p < 0.05), of which chromosome-type aberrations (43.2 +/- 10.3 on 100 metaphases) averaged 92.5%. In lymphocytes of 37 out of 43 HL antitumoral treatment patients, we found, in addition to ordinary aberrant cells, a large number of multiaberrant (MA-cells) cells, i.e. metaphases carrying multiple (at least four) chromosome-type exchange aberrations. In 30 non-treated HL patients only one MA-cell was found. From 171 MA-cells which were in 43 HL patients after antitumoral treatment, 114 MA-cells were found at inspection of 9766 diploid metaphases, and the remaining 57 MA-cells were found at inspection of 196 polyploid metaphases. The carrying out after chemotherapy of radiation therapy enlarges in lymphocytes frequency of appearance of MA-cells. The analysis of MA-cells in diploid and polyploid metaphases shown, that the MA-cells could be formed both in vivo, and in vitro in absence of influence of clastogenic factors, and could survive at least two rounds of in vitro replication.     

The database for analysis of quantitative characteristics of chromosome aberration frequencies in the culture of human peripheral blood lymphocytes]

The data on spontaneous chromosome aberration rates in cultures of human peripheral blood lymphocytes obtained in the past 30 years have been collected to form a database. The database contains the results of analysis of more than 330,000 metaphases in lymphocytes from more than 1200 subjects. The frequency of aberrant metaphases in the control group has been estimated at 0.0213 +/- 0.00085. No differences between sexes have been found with respect to either the total chromosome aberration rate or the rates of individual aberration types. The total chromosome aberration rate did not depend on age; however, it has been found that the number of fragments increased and the number of exchanges decreased with age. Smoking has been found to increase the frequency of chromosome aberrations in individuals with occupational hazards, but not in those who are not occupationally exposed to radiation or chemicals. Alcohol consumption increased the frequency of paired fragments, whereas the frequencies of other aberrations did not differ from the control values.     

The Database for Analysis of Quantitative Characteristics of Chromosome Aberration Frequencies in the Culture of Human Peripheral Blood Lymphocytes

The data on spontaneous chromosome aberration rates in cultures of human peripheral blood lymphocytes obtained in the past 30 years have been collected to form a database. The database contains the results of analysis of more than 330 000 metaphases in lymphocytes from more than 1200 subjects. The frequency of aberrant metaphases in the control group has been estimated at 0.0213 ± 0.00085. No differences between sexes have been found with respect to either the total chromosome aberration rate or the rates of individual aberration types. The total chromosome aberration rate did not depend on age; however, it has been found that the number of fragments increased and the number of exchanges decreased with age. Smoking has been found to increase the frequency of chromosome aberrations in individuals with occupational hazards, but not in those who are not occupationally exposed to radiation or chemicals. Alcohol consumption increased the frequency of paired fragments, whereas the frequencies of other aberrations did not differ from the control values.     

The effect of a Coxsackie viral infection on the chromosomal apparatus of the cells of fetuses and newborn mice]

The Coxsackie virus B1 has been studied for its effect on the chromosome apparatus in cells of mouse feti and newborn mice in case of intraperitoneal infection of females on the 3d week of pregnancy. It is stated that the virus readily moves across the placental barrier and induces chromosome aberrations in the cells of feti, their number being 2.5 times higher than the control level. The maximal number of structural chromosome aberrations was registered during the first three days from the moment of infecting. Then a gradual decrease in the number of aberrant metaphases in the cells of newborn mice was observed. It had reached the value of the control level by the 17th day of observation.     

Cell survival and radiation induced chromosome aberrations

Human peripheral lymphocytes were irradiated in whole blood with 0.5-4.0 Gy of 220 kVp X-rays and the frequency of chromosome aberrations was determined in 1st or 2nd division metaphases discriminated by fluorescence plus giemsa staining. Using the empirical distributions of aberrations among cells, cell survival and transmission of aberrations were investigated. Considering both daughter cells, we found that 20% of fragments and 55% of dicentrics or ring chromosomes are lost during the 1st cell division; i.e. cell survival rate from 1st to 2nd generation is mainly influenced by anaphase bridging of these two-hit aberrations. Cell survival to 2nd mitosis was calculated considering this situation and compared with the survival derived from the fraction of M 1 cells without unstable aberrations. The resulting shouldered survival curves showed significantly different slopes, indicating that cell reproductive death is overestimated in the latter approach.     

Blood lymphocyte culture system: quantitative analysis of X-ray-induced chromosome aberrations in man, muntjac and cattle

Peripheral blood lymphocytes of 3 mammalian species, man, muntjac and cattle, which have various amounts of DNA and divergent karyotypes, were exposed to 100-400 rad of X-rays, and frequencies of dicentrics and other aberrations were analysed at first post-irradiation metaphases. During experiments, various preparative or physical and biological factors that could influence the yield of chromosome aberrations were taken into account. The frequency of dicentrics scored at first post-irradiation metaphases showed best fit to both linear and quadratic dose-response curves, y = a + bD and y = bD + cD2 with a high correlation coefficient of 0.98 (P less than 0.001). The frequency of dicentrics obtained at different post-irradiation fixation times did not show significant variation, indicating a homogeneous sensitivity of peripheral lymphocytes to X-irradiation. BrdU incorporation following X-irradiation showed no increase in the frequency of chromosome aberrations. The frequency of dicentrics in man, muntjac and cattle showed a close correlation with their DNA content, but no meaningful correlation was found between the yield of dicentrics and the chromosome arm number or the nuclear volume. The ratio of dicentric yields, 1.00:0.67:1.04 obtained in man, muntjac and cattle were comparable to the ratio of their DNA contents, 1.00: 0.65: 1.07. The base-line frequency of SCEs was similar in the 3 species and no significant variation in SCE frequency was noticed even after administration of 400 rad of X-rays.     

Chromosome radiosensitivity in 2 successive mitotic cycles of human lymphocytes

A culture of human lymphocytes was irradiated with gamma-quanta (0.5 Gy) after incubation during different time intervals, i.e. at different relative frequencies of cells in the first and second mitotic cycle. The frequency of aberrations produced in the G2 stage was analysed. The total yield of aberrations gradually increased with the increase of time lapse between the onset of cultivation and irradiation. If, however, the relative frequencies of the first and second mitoses are taken into account, it could be concluded that radiosensitivity of chromosomes remains constant in each cycle and independent from its duration. Besides, radiosensitivity of chromosomes during the second cycle is one and half times stronger, as compared to the first cycle. In accordance with the data of other authors, 5-bromodeoxyuridine has been found to significantly increase radiosensitivity.     

Trenimon-induced SCEs and structural chromosomal aberrations in early- and late-dividing lymphocytes

In cultures of human peripheral lymphocytes the frequencies of Trenimon-induced SCEs in second post-treatment metaphases and of structural chromosomal aberrations in first, second and third post-treatment metaphases were clearly lower at late as compared with early fixation times. These results, which are discussed, indicate that T cells (early dividing) are more sensitive with respect to the induction of SCEs and structural chromosomal aberrations by Trenimon as compared with B cells (late dividing).     

Chromosome aberrations in peripheral lymphocytes and radiation dose to active bone marrow in patients treated for cancer of the cervix

An international study of cervical cancer patients reported a doubling of the risk for leukemia following radiotherapy. To evaluate the extent of residual chromosome damage in circulating T-cell lymphocytes in this population, approximately 200 metaphases were examined from each of 96 irradiated and 26 nonirradiated cervical cancer patients treated more than 17 years ago (average 23 years). Radiation dose averaged over the total red bone marrow was estimated to be 8.1 Gy. The type and frequency of stable and unstable chromosome aberrations were quantified in 24,117 metaphases. Unstable aberrations did not differ significantly between irradiated and nonirradiated patients (P greater than 0.5). Stable aberrations (i.e., translocations, inversions, or chromosomes with deleted segments), however, were significantly higher among irradiated (2.8 per 100 cells) compared to nonirradiated (0.7 per 100 cells) women (P less than 10(4). The frequency of these stable aberrations was found to increase significantly with increasing dose to the bone marrow. These data indicate that a direct relationship between radiation dose and extent of damage to somatic cells persists in populations and can be detected many years after partial-body radiation exposure. The stable aberration rate in irradiated cervical cancer patients was 50 to 75% lower than those observed 25 years or more after radiation exposure in atomic bomb survivors and in ankylosing spondylitis patients treated with radiotherapy. The average marrow dose was only 1 Gy in the examined atomic bomb survivors and 3.5 Gy in the ankylosing spondylitis patients. It appears, then, that a very high dose delivered to the pelvic cavity in fractionated doses resulted in far fewer persistent stable aberrations than lower doses delivered either in acute whole-body exposure or in fractionated doses to the spinal column and sacroiliac joints. The higher radiation dose and the concentration of that dose in a smaller area of the body appear to be responsible for the lower rate of persistent aberrations observed in cervical cancer patients.     

Frequency and types of induced and spontaneous chromosome aberrations in relation to cell kinetics

Summary Induced and spontaneous structural chromosome aberrations (SCA) were studied in a child accidentally radiated with a high dose of ¹⁹²Ir, and in three sibs with Fanconi's anemia, analyzing by separate first division metaphases (FDM) and second division metaphases (SDM). The results showed that the number of SCA, number of cells with aberrations, and SCA per cell were markedly higher in FDM in all patients.Furthermore, for some type of structural changes like dicentric chromosomes and chromatid interchanges, the differences were particularly striking. The importance of ascertaining FDM identified with proper techniques, for the study of the clastogenic effect of environmental agents and some aspects related to the differences in cytogenetic features found in diverse tissues in Fanconi's anemia are discussed.     

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.     

A comparison of aberration distribution and cell-cycle progression in cells treated with bleomycin with those exposed to X-rays

The extent of cell-cycle delay and the frequency of aberrant metaphases induced by bleomycin (BLM) and X-rays have been compared at doses which produce similar frequencies of chromosome aberrations by the 2 clastogenic agents (BLM, 40 micrograms/ml and X-rays, 2 Gy) in muntjac lymphocytes. The frequency of aberrant metaphases was low in BLM-treated cells; however, the number of aberrations per metaphase was higher than in cells exposed to X-rays. Thus in contrast to their uniform sensitivity to X-rays, the lymphocytes showed differential sensitivity to BLM. This might be due to differences among the cells in their uptake of BLM and/or its action on the nuclear membrane-DNA complex. In spite of the total number of chromosome aberrations being similar to that induced by X-rays, BLM did not induce a significant delay in cell-cycle progression as observed in the case of X-rays. A possible explanation could be that the DNA damages being limited to fewer cells than in the case of X-irradiation, the BLM-treated cultures had more normal cells allowing faster progression and/or unlike X-rays BLM may not be causing other cellular damages in addition to DNA breaks.     

Anticlastogenic effect of Vitamin C on cisplatin induced chromosome aberrations in human lymphocyte cultures.

Vitamin C (ascorbic acid) is an antioxidant that can scavenge free radicals and protect cellular macromolecules, including DNA, from oxidative damage induced by different agents. The protective effect of Vitamin C on cisplatin induced chromosome aberrations has been determined in the human peripheral lymphocyte chromosome aberration test in vitro. The results of treatments with Vitamin C indicated that it statistically significantly decreases the number of chromosome aberrations and number of metaphases with aberrations induced with cisplatin, but it can not completely protect cells from damage. The test concentrations of Vitamin C (10 and 100 microg/ml) had a limited antimutagen effect on cisplatin (0.5 microg/ml), which can cause genetic damage through free radical mechanisms. The antimutagen effect included the anticlastogenic effect of Vitamin C and its ability to decrease the number of aneuploid mitoses. Vitamin C showed the most efficient anticlastogenic effect during simultaneous treatment with cisplatin. Also, Vitamin C reduced cell toxicity of cisplatin during simultaneous treatment.     

A comparative study of the micronucleus test and chromosome aberrations in PHA-stimulated human lymphocytes

A dose dependence of the number of cells with chromosome aberrations was studied in PHA-stimulated donor's peripheral blood lymphocytes irradiated in vitro with doses of 10-400 cGy. In studying the number of chromosome aberrations and percentage of cells with micronuclei in parallel cultures no correlation was found between these indices within the groups exposed to a similar radiation dose.     

Chromosome and cytome analysis of the somatic cells of nuclear-chemical production workers with incorporated 239Pu

The analysis of plutonium production factors has been carried out by using two methodical approaches: assessment of chromosomal aberrations level in routine and G-banded metaphases and molecular-cytogenetic investigation of aneugenic/clastogenic damages in cytokinesis-block binuclear lymphocytes by FISH with centromere specific DNA probes. The obtaining data point out for the first time about both aneugenic and clastogenic influences of incorporated 239Pu with activity range from 0.37 to 6.95 kBq. Correlation analysis of chromosome aberrations with cytome abnormalities allowed finding significant connection between number parameters of metaphase and interphase approaches. The results of this study support the suggestion that aberrant chromosomes are involved preferable in aneugenic events. The FISH technique in binucleated cytokinesis-blocked lymphocytes allows extending of detecting spectrum of chromosome damages and glance of aneugenic mechanisms. Correlations between metaphase and interphase-FISH results point out a high sensitivity of FISH cytome assay, which could be used as an independent test for detection both clastogenic and aneugenic environment influences.     

Direct analysis of the clastogenic effect of formaldehyde in unstimulated human lymphocytes by means of the premature chromosome condensation technique

The cytogenetic effect of formaldehyde (FA) on unstimulated human lymphocytes was studied by means of conventional chromosome analysis and the premature chromosome condensation (PCC) technique. In first post-treatment metaphases no significantly increased yields of chromosomal changes could be observed. The analysis of PCCs, however, showed high yields of chromosome fragments. Bleomycin (BLM) used as positive control was also highly clastogenic in PCCs and resulted in significantly increased yields of chromosome-type aberrations. As recently argued, a premitotic selection against heavily damaged cells could be an explanation for the discrepancy between the chromosome findings in metaphase and PCC analysis after FA treatment. In addition, a differential effectiveness may exist in unstimulated lymphocytes to convert multiple fragmentation into chromatid- or chromosome-type aberrations through S-phase-dependent or S-phase-independent mechanisms.     

Aberrant and multiaberrant (rogue) cells in peripheral lymphocytes of Hodgkin's lymphoma patients after chemotherapy

We analyzed spontaneous chromosome lesions in peripheral lymphocytes cultured from Hodgkin's lymphoma (HL) patients before and after cytostatic chemotherapy. The mean aberration frequency was significantly higher in HL patients after chemotherapy (7.20+/-0.58 per 100 metaphases) than in non-treated HL patients (4.80+/-0.54), and in non-treated patients than in healthy subjects (2.12+/-0.13). In lymphocytes of HL patients, who received chemotherapy, we found, in addition to ordinary aberrant cells, a large number of multiaberrant (or rogue) cells, i.e. metaphases carrying multiple (at least four) chromosome-type exchange aberrations. Rogue cells were found in 15 out of 18 chemotherapeutically treated HL patients (in total, 60 rogue cells per 5,568 scored cells), whereas in 30 non-treated patients only 1 rogue cell was found (per 4,988 scored cells). No correlation was found between the yield of rogue cells and the aberration frequency in ordinary aberrant cells. Aberration spectra (ratios of chromatid- to chromosome-type aberrations and of breaks to exchanges) were essentially different in ordinary aberrant and multiaberrant cells. These data, as well as analysis of cellular distributions of aberrations, implied independent induction of chromosome damage in ordinary aberrant and rogue cells. Analysis of aberration patterns in diploid and polyploid rogue metaphases belonging to the first, second, and third in vitro division indicated that rogue cells could be formed both in vivo and in vitro, and could survive at least two rounds of in vitro replication, given blocked chromosome segregation. These results suggested that formation of rogue cells, unlike ordinary aberrant cells, was triggered by events other than direct DNA and/or chromosome lesions. A hypothesis regarding disrupted apoptosis as a candidate mechanism for rogue cell formation seems to be most suitable for interpretation of our data. Cultured lymphocytes of chemotherapeutically treated HL patients may represent a model system for further examination of the multiaberrancy phenomenon.