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.     

Micronuclei in human lymphocytes: the Co-60 gamma-ray dose-response

Dose-response for micronuclei in cytokinesis-blocked lymphocytes after in vitro irradiation of whole blood from 3 donors with Co-60 gamma-rays in the range 0–5.0 Gy was established. The numerical relationship between radiation induced chromosomal aberrations, and micronuclei is also examined. An increased frequency of micronuclei following low doses of gamma-irradiation is reported from a study of 41 radiation workers.     

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.     

Micronuclei in lymphocytes of prostate cancer patients undergoing radiation therapy

To further verify the applicability of the micronucleus (MN) assay in biodosimetry, we measured the MN yield in cytokinesis-blocked (CB) peripheral blood lymphocytes (PBL) of eight prostate cancer (PC) patients. These patients had no previous chemotherapy or radiotherapy (xRT). They were treated with standardized schemes of fractionated pelvic xRT. Before xRT, and at one random time-point during the course of xRT, blood samples were collected from each patient for the following purposes: (1) to verify the relationship between the MN yield in PBL and the estimated equivalent (EQ) total-body absorbed dose; and (2) to evaluate the individual differences of ex vivo radiation dose-response (1-4 Gy) relationship of MN yield in PBL before xRT. The number of xRT fractions, cumulative tumor dose, and EQ total-body absorbed doses of these patients represented a wide range. We found in PBL of these patients that (1) MN yield (Y) increased linearly with the estimated EQ total-body absorbed dose as Y=14.6+9.2D (R(2)=0.7, p=0.007); the distributions of MN yield were overdispersed; the ratio of relative increment of MN yield per 1000 binucleated (BN) PBL ranged from 0.9 to 8.2 (median: 4.1) folds above that of the respective baseline levels; and (2) before xRT, the MN yields also increased linearly with the ex vivo radiation dose; at each radiation dose level, the distributions of MN yield were overdispersed in most patients. In two of the three patients with xRT-induced early side effects (cystitis, diarrhea), the MN yield in PBL induced by ex vivo irradiation before xRT was significantly higher than in the other patients without xRT-induced side effects. These findings suggest that MN yields in CB PBL can be used as an in vivo biodosimeter. Since the differences in individual ex vivo radiation dose-response relationship of MN yield in PBL before xRT appeared to be significant, our preliminary results also suggest that it may be possible to identify individual intrinsic radiosensitivity before the start of xRT.     

The induction of micronuclei in human lymphocytes by low doses of radiation

The appearance of micronuclei (MN) is delayed with respect to cell division in populations of irradiated human lymphocytes, so that the length of time in culture, as well as the number of divisions, is a factor in MN assays. Using two assays that control for cell kinetics, we measured the yield of cells with MN exposed to graded doses of 60Co gamma rays and 90KVP X-rays. The yields showed a non-linear increase with dose. They can be represented by two straight lines: the one in the range below 0.15 Gy has a slight slope, the other in the range above 0.15 Gy has a significantly greater slope. The radical scavengers cysteamine and glycerol, which reduced the MN yields sharply at 3 Gy, were less effective at 0.3 Gy, indicating that terminal deletions arising from the direct ionization of DNA are a major source of the MN induced by low radiation doses. It is likely that the non-linear dose response is due to the saturation of a DNA repair process.     

Cytogenetic monitoring of industrial radiographers using the micronucleus assay

Industrial radiography is the process of using either gamma-emitting radionuclide sources or X-ray machines to examine the safety of industrial materials. Industrial radiographers are among the radiation workers who receive the highest individual occupational radiation doses. To assess occupationally induced chromosomal damage, we performed the cytokinesis-block micronucleus (CBMN) assay in peripheral lymphocytes of 29 male industrial radiographers, exposed to ionizing radiation for 12.8 years±11.2, in comparison with 24 gender-, age-, and smoking habits-matched controls. The CBMN assay was combined with fluorescent in situ hybridization with a pan-centromeric DNA probe in 17 exposed subjects and 17 controls randomized from the initial populations. The mean cumulative equivalent dose, recorded by film dosimeters, was 67.2 mSv±49.8 over the past 5 years. The mean micronucleated binucleated cell rate (MCR) was significantly higher in the industrial radiographers than in the controls (10.7‰±5.2 versus 6.6‰±3.1, P=0.009); this difference was due to a significantly higher frequency of centromere-negative micronuclei (C−MN) in exposed subjects than in controls (8.5‰±4.9 versus 2.2‰±1.6, P<0.001). The two populations did not significantly differ in centromere-positive micronuclei (C+MN) frequency. These findings demonstrate a clastogenic effect in lymphocytes of industrial radiographers. MCR significantly positively correlated with age in the two groups. After correction for the age effect, MCR did not correlate with duration of occupational exposure. No correlation between radiation doses and MCR, C−MN, and C+MN frequencies was observed. In addition to physical dosimetry records, the enhanced chromosomal damage in lymphocytes of industrial radiographers emphasizes the importance of radiation safety programs.     

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.     

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.     

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.     

Induction of micronuclei by X-radiation in human, mouse and rat peripheral blood lymphocytes

We compared the radiosensitivity of human, rat and mouse peripheral blood lymphocytes (PBLs) by analyzing micronuclei (MN) in cytochalasin B-induced binucleated (BN) cells. For each species and dose 4-ml aliquots of whole blood were X-irradiated to obtain doses of 38, 75, 150 or 300 cGy. Controls were sham-irradiated. After exposure to X-rays, mononuclear leukocytes were isolated using density gradients and cultured in RPMI 1640 medium containing phytohemagglutinin to stimulate mitogenesis. At 21 h cytochalasin B was added to produce BN PBLs, and all cultures were harvested at 52 h post-initiation using a cytocentrifuge. Significant dose-dependent increases in the percentage of micronucleated cells and the number of MN per BN cell were observed in all three species. The linear-quadratic regression curves for the total percentage of micronucleated cells for the three species were similar; however, the curve for the mouse PBLs had a larger quadratic component than either of the curves for the rat or human PBLs. Although the correlation between the percentage of cells with MN and those with chromosome aberrations was high (r2 greater than 0.95), the mouse and rat PBLs were over twice as efficient as human PBLs in forming MN from presumed acentric fragments. These data indicate that the induction of MN in BN cells following ionizing radiation is similar in human, rat and mouse PBLs, but care must be taken in using the MN results to predict frequencies of cells with chromosomal aberrations.     

Relationships between satellite association and the occurrence of non-disjunction in man.

It has been reported that there is an increased incidence of Down's syndrome among the children of parents who have been exposed to ionizing radiations for radiodiagnostic or radiotherapeutic reasons. Work with Drosophila, mice and human lymphocytes has shown that irradiation with X- or gamma-rays induces aneuploidy, presumably by non-disjunction. It has been suggested that in man the frequency of satellite association (s.a.) of acrocentric chromosomes may be involved in the causation of chromosomal non-disjunction. In the present work the effects of radiation on s.a. have, therefore, been investigated. The frequency of s.a. between acrocentric chromosomes was determined after the exposure of human blood from normal and chromosomally abnormal individuals to various small doses of Co-60 gamma-rays. The criteria of Zang and Back were used for the evaluation of s.a. complexes. No effects of radiation on the frequency of s.a. were apparent within the dose range investigated. The same result was obtained when s.a. was evaluated using the silver-staining technique in which physical connections between the associating satellites may be observed and the association complexes evaluated directly. The effects of other radiation sources have also been investigated.     

Cell killing, nuclear damage and apoptosis in Chinese hamster V79 cells after irradiation with heavy-ion beams of (16)O, (12)C and (7)Li

Chinese hamster V79 cells were exposed to high LET (linear energy transfer) (16)O-beam (625keV/mum) radiation in the dose range of 0-9.83Gy. Cell survival, micronuclei (MN), chromosomal aberrations (CA) and induction of apoptosis were studied as a follow up of our earlier study on high LET radiations ((7)Li-beam of 60keV/mum and (12)C-beam of 295keV/mum) as well as (60)Co gamma-rays. Dose dependent decline in surviving fraction was noticed along with the increase of MN frequency, CA frequency as well as percentage of apoptosis as detected by nuclear fragmentation assay. The relative intensity of DNA ladder, which is a useful marker for the determination of the extent of apoptosis induction, was also increased in a dose dependent manner. Additionally, expression of tyrosine kinase lck-1 gene, which plays an important role in response to ionizing radiation induced apoptosis, was increased with the increase of radiation doses and also with incubation time. The present study showed that all the high LET radiations were generally more effective in cell killing and inflicting other cytogenetic damages than that of low LET gamma-rays. The dose response curves revealed that (7)Li-beam was most effective in cell killing as well as inducing other nuclear damages followed by (12)C, (16)O and (60)Co gamma-rays, in that order. The result of this study may have some application in biological dosimetry for assessment of genotoxicity in heavy ion exposed subjects and in determining suitable doses for radiotherapy in cancer patients where various species of heavy ions are now being generally used.     

Cytogenetic monitoring of industrial radiographers using the micronucleus assay

Industrial radiography is the process of using either gamma-emitting radionuclide sources or X-ray machines to examine the safety of industrial materials. Industrial radiographers are among the radiation workers who receive the highest individual occupational radiation doses. To assess occupationally induced chromosomal damage, we performed the cytokinesis-block micronucleus (CBMN) assay in peripheral lymphocytes of 29 male industrial radiographers, exposed to ionizing radiation for 12.8 years+/-11.2, in comparison with 24 gender-, age-, and smoking habits-matched controls. The CBMN assay was combined with fluorescent in situ hybridization with a pan-centromeric DNA probe in 17 exposed subjects and 17 controls randomized from the initial populations. The mean cumulative equivalent dose, recorded by film dosimeters, was 67.2 mSv+/-49.8 over the past 5 years. The mean micronucleated binucleated cell rate (MCR) was significantly higher in the industrial radiographers than in the controls (10.7 per thousand +/-5.2 versus 6.6 per thousand +/-3.1, P=0.009); this difference was due to a significantly higher frequency of centromere-negative micronuclei (C-MN) in exposed subjects than in controls (8.5 per thousand +/-4.9 versus 2.2 per thousand +/-1.6, P<0.001). The two populations did not significantly differ in centromere-positive micronuclei (C+MN) frequency. These findings demonstrate a clastogenic effect in lymphocytes of industrial radiographers. MCR significantly positively correlated with age in the two groups. After correction for the age effect, MCR did not correlate with duration of occupational exposure. No correlation between radiation doses and MCR, C-MN, and C+MN frequencies was observed. In addition to physical dosimetry records, the enhanced chromosomal damage in lymphocytes of industrial radiographers emphasizes the importance of radiation safety programs.     

Cytogenetic damage induced in human lymphocytes by sodium bisulfite.

The frequencies of chromosomal aberrations (CA), sister-chromatid exchanges (SCE), and micronuclei (MN) in human blood lymphocytes exposed to sodium bisulfite (sulfur dioxide) at various concentrations ranging from 5 x 10(-5) M to 2 x 10(-3) M in vitro were studied. It was shown that sodium bisulfite (NaHSO3 and Na2SO3, 1:3 M/M) caused an increase in SCE and MN in human blood lymphocytes in a dose-dependent manner, and also induced mitotic delays and decreased mitotic index. For CA, our results indicated that sodium bisulfite induced an increase of chromatid-type aberrations in lymphocytes from three of four donors in a dose-dependent manner. The chemical at low concentrations induced chromatid-type aberrations, but not chromosome-type aberrations; high concentrations induced both chromatid- and chromosome-type aberrations. No cytogenetic damage in human lymphocytes was induced by sodium sulfate. The results have confirmed that sulfur dioxide is a clastogenic and genotoxic agent.     

Micronucleus frequency is increased in peripheral blood lymphocytes of nuclear power plant workers

Nuclear power plant workers are exposed to ionizing radiation at relatively low doses and for prolonged periods of time. To investigate the extent of genetic damage in these workers, a group of 133 nuclear power plant workers and 39 healthy controls were compared using the cytokinesis-block micronucleus assay. The frequency of micronuclei was significantly increased in peripheral lymphocytes of nuclear power plant workers (20.5 +/- 9.7% compared to 13.7 +/- 5.9%). A significant dose-response relationship was observed between micronucleus (MN) frequency and both the accumulated dose and the duration of employment (P < 0.01 for both variables after adjusting for age, gender and cigarette smoking) with an evident leveling off for exposures over 200 mSv. Accumulated dose and duration of employment were significantly correlated but exerted independent effects on MN frequency. For non-occupational parameters, age was significantly associated with the frequency of micronuclei, while gender was not. Smoking habit showed no overall effect, whereas increased chromosome damage was evident in smokers of more than 20 cigarettes per day. In conclusion, a dose-related association between MN frequency and exposure to ionizing radiation was evident in nuclear power plant workers, encouraging the application of the cytokinesis-block micronucleus assay in biomonitoring studies of human populations with prolonged exposure to ionizing radiation.     

Ginseng reduces the micronuclei yield in lymphocytes after irradiation

To assess the effect of Chinese ginseng in modifying the radiation-induced micronuclei (MN) yield in human G(o) peripheral blood lymphocytes (PBL), we conducted the cytokinesis-blocked (CB) MN assay in blood samples obtained from healthy volunteers (n=4). Before (137)Cs ex vivo irradiation, mononuclear cell cultures from each sample were incubated 24 h with different concentrations (0-2000 microg ml(-1)) of crude water extract of ginseng dry root. We found that (1) at 0 Gy and without the presence of ginseng, MN yield (mean+/-S.E.M.) was 11.7+/-2.7 per 1000 binucleated (BN) cells. Different concentrations of ginseng crude water extract did not affect the MN yields and the proliferative activity of PBL; (2) after 1 and 2 Gy exposure, radiation alone sharply increased the MN yields, respectively, to 119.6+/-17.4 and 340.5+/-20.9 per 1000 BN cells. However, treatment with ginseng for 24 h before radiation exposure, resulted in a significant linear decline of MN yields as ginseng concentration increases. Compared to radiation alone, the extent to which ginseng water extract reduced the MN yields induced by 1 Gy exposure was 46.0% at 1500 microg ml(-1) and 61.5% at 2000 microg ml(-1), and with 2 Gy exposure, it was 38.6% at 1500 microg ml(-1) and 46.5% at 2000 microg ml(-1); (3) MN data suggested a tendency for overdispersion relative to the Poisson model; and (4) over the different levels of ginseng concentrations, the trend in micronucleated BN index was as similar as that of the MN yields. These results indicated that (1) ginseng crude water extract exerts no apparent cytogentic effect on human PBL at concentrations up to 2000 microg ml(-1) as evaluated by the CBMN assay; and (2) the protection of ginseng water extract against (137)Cs-induced MN in human PBL is concentration-dependence. Therefore, our findings indicated that ginseng may have therapeutic value as a possible radioprotector for normal tissue during radiotherapy of cancer patients.     

Induction of micronuclei by 153Sm-EDTMP in peripheral blood lymphocytes of patients with bone metastases.

The purpose of this study was to evaluate the degree of cytological radiation damage to peripheral blood lymphocytes induced by 153Sm-EDTMP applied for palliation of metastatic bone pain. Blood samples from 16 patients (46-82 years old), 10 without previous radiotherapy and 6 with previous radiotherapy, were collected before and one hour after the administration of a mean activity of 41.7+/-5.8 MBq/kg of 153Sm-EDTMP. Then the lymphocytes were cultured for cytokinesis block micronucleus (MN) assay. The number of MNper binucleated cells (BC) in patients without previous radiotherapy before the treatment was of 0.030 (+/- 0.016) and after one hour 0.035 (+/- 0.013), although we could find inter individual differences. The basal MN/BC of the patients with no previous radiotherapy was similar to the controls. The increment in the percentage of BC with MN was similar in patients with and without previous radiotherapy. The observed mean of MN/BC is equivalent to a dose range of 0.05 to 0.10 Gy of 153Sm-EDTMP in vitro. The relatively low frequency of lymphocyte with micronuclei after the exposure to 153Sm-EDTMP supported the contention that radiation damage in lymphocytes of patients with painful bone metastases is minimal.     

Frequency of radiation-induced micronuclei in neuronal cells does not correlate with clonogenic survival

It is generally assumed that radiation-induced micronuclei (MN) in cytokinesis-blocked cells are an expression of cellular radiosensitivity. Therefore, radiosensitive cells should have a high frequency of MN and radioresistant cells should show lower levels. We have irradiated cells of a panel of 13 neuronal cell lines of widely differing radiosensitivity [human neuroblastomas: N2alpha, SHSY5Y, SK-N-SH, KELLY and SK-N-BE(2c); murine neuroblastomas: OP-6 and OP-27; human glioblastomas: G120, G60, G28, G112, G44 and G62] and compared their radiation response using the micronucleus and standard clonogenic assays. It was found that micronucleus frequency was much higher in some of the radioresistant cell lines (N2alpha, G28, G120 and G44; SF2 >/= 0.60). These cell lines showed a high frequency of more than 0.32 MN per gray of (60)Co gamma radiation per binucleated cell. On the other hand, the more radiosensitive cell lines (OP-27 and SK-N-SH, SF2 相似文献   

Adaptive response of blood lymphocytes of the inhabitants of the South Ural chronically exposed to radiation

The inhabitants of Techa river villages exposed to injured radiation action and inhabitants of uncontaminated regions were examined by micronuclei (MN) assay. The initial damage of blood lymphocytes, the role of radiation in the induction of sensitivity to the acute irradiation and the ability to form the adaptive response were evaluated. It was shown that the initial level of damaged lymphocytes in the inhabitants of the contaminated area did not differ significantly from the spontaneous level. But in these people the sensitivity to acute irradiation was decreased. The ability to develop the adaptive response was decreased too. It was suggested that the radiation plays the main role in the development of radiosensitivity and adaptive response in chronically irradiated people.     

Quantitative analyses of the induction of chromosome aberrations and sister-chromatid exchanges in human lymphocytes exposed to gamma-rays and mitomycin-C in combination

Most chemicals are S-dependent and are potent inducers of SCE, but do not produce chromosome-type aberrations in the first metaphases after exposure. Ionizing radiation, which is an S-independent agent, produces chromosome-type aberrations, especially dicentrics and rings, but inefficiently produces chromatid-type aberrations. A series of experiments has been performed to investigate whether cytogenetic damage induced by ionizing radiation (gamma-rays) might be assessed separately from that induced by the alkylating chemical, mitomycin C (MMC), when human lymphocytes were exposed to these 2 agents in combination. Whole-blood cultures of human lymphocytes in G0 phase were exposed to gamma-rays and MMC in combination or separately. Cytogenetic analyses were done for both chromosome aberrations (CA), analyzed in cultures incubated for 56 h without BrdUrd, and sister-chromatid exchanges (SCEs) in cultures incubated for 72 h with BrdUrd. The frequency of chromosome-type aberrations (dicentrics and rings) increased with increasing doses of gamma-rays from 0.5 to 4.0 Gy. The dose-response relationships were the same with or without concomitant treatment with MMC (10(-6) M). Although the SCE frequency increased with increasing doses of MMC, the increase was nearly the same as when cells were treated with both MMC and gamma-rays (2 Gy). There was no interaction between MMC and gamma-rays concerning these 2 endpoints.