Mentha piperita (Linn.) leaf extract provides protection against radiation induced chromosomal damage in bone marrow of mice

Oral administration of M. piperita (1 g/kg body weight/day) before exposure to gamma radiation was found to be effective in protecting against the chromosomal damage in bone marrow of Swiss albino mice. Animals exposed to 8 Gy gamma radiation showed chromosomal aberrations in the form of chromatid breaks, chromosome breaks, centric rings, dicentrics, exchanges and acentric fragments. There was a significant increase in the frequency of aberrant cells at 6 hr after irradiation. Maximum aberrant cells were observed at 12 hr post-irradiation autopsy time. Further, the frequency of aberrant cells showed decline at late post-irradiation autopsy time. However, in the animals pretreated with Mentha extract, there was a significant decrease in the frequency of aberrant cells as compared to the irradiated control. Also significant increase in percentage of chromatid breaks, chromosome breaks, centric rings, dicentrics, exchanges, acentric fragments, total aberrations and aberrations/damaged cell was observed at 12 hr post-irradiation autopsy time in control animals, whereas Mentha pretreated irradiated animals showed a significant decrease in percentage of such aberrations. A significant decrease in GSH content and increase in LPO level was observed in control animals, whereas Mentha pretreated irradiated animals exhibited a significant increase in GSH content and decrease in LPO level but the values remained below the normal. The radioprotective effect of Mentha was also demonstrated by determining the LD(50/30) values (DRF = 1.78). The results from the present study suggest that Mentha pretreatment provides protection against radiation induced chromosomal damage in bone marrow of Swiss albino mice.     

Chromosome aberration peristence in swine leukocytes following exposure to fission neutrons

Chromosome aberration rates in swine lymphocytes decreased rapidly within the first 24 h postirradiation. This initial loss coincided with a decrease in lymphocyte number and continued until lymphocyte numbers began to recover at 7 days after irradiation. Subsequent losses were less dramatic, and numbers of acentric fragments reached preirradiation levels by 56 days postexposure; rings and dicentrics persisted throughout the 84-day study. Mathematical models descrine the predicted aberration yield as a function of time after irradiation. The disappearance rate of aberrant lymphocytes after irradiation suggests that anerration persistence kinetics should be considered when using lymphocyte aberrations as biologic dosimeters.     

Influence of mitotic delay on the results of biological dosimetry for high doses of ionizing radiation

The purpose of this study was to systematically investigate how high doses of sparsely and densely ionizing radiations influence the proliferation time of lymphocytes in short-term cultures and, consequently, the observed frequencies of dicentric and centric ring chromosomes. Peripheral blood samples from five volunteers were irradiated with high doses of 200 kV X-rays and with neutrons with a mean energy of <E _n>=2.1 MeV. First division metaphase cells were collected after different culture times of 48, 56, and 72 h and dicentrics, centric ring chromosomes, and acentric fragments were determined. The data hint at considerable mitotic delay. The main increase in the number of chromosome aberrations occurred between 48 and 72 h after an X-ray exposure and between 56 and 72 h after neutron exposure. When the data were used for a calibration of aberration frequency versus dose, subsequent dose estimations resulted, however, in comparable values. Thus, in spite of the influence of mitotic delay on observable chromosome aberrations, at least for the radiation types investigated here, a culture time of 48 h is acceptable for biological dosimetry.     

Radiation-induced chromosomal aberrations in the bone marrow of mice exposed to various doses of gamma radiation

The occurrence of chromosomal aberrations was studied at 1–14 days post-exposure in female BALB/c mice exposed to various doses of gamma radiation. The frequency of abnormal cells, chromatid and chromosome breaks, dicentrics, centric rings, acentric fragments and total aberrations increased with exposure dose, and it was highest at 7 Gy. A peak was recorded on day 1 post-exposure with a gradual decline thereafter. The chromosomal aberration yield reached a nadir on day 14 post-irradiation, without restoration to the control level. The best fit for the present data was by a linear-quadratic relationship between dose of radiation and the frequency of chromosomal aberrations.     

Cytogenetic effects induced in vitro in human peripheral blood lymphocytes by neutrons. II. Relative biological effectiveness of neutrons having different energies]

Human lymphocytes were irradiated in vitro during Go stage by graded doses of thermal neutrons and neutrons having an average energy of 0.04; 0.09; 0.35; 0.85 and 14,7 MeV as well as by 60Co gamma rays, and RBE of neutrons relative to gamma-rays was calculated for the frequency of total and different types of aberrations. It was found that the RBE has the most value at the low doses and decreases when the exposition dose increases. 0.35 MeV neutrons have the maximum RBE in comparison with neutrons having other energies. When comparing the RBE values calculated for different types of chromosome aberrations, it was found out that dicentrics and dicentrics plus centric rings had more RBE than acentric aberrations (pair fragments and minutes).     

In vitro transmission of chromosomal aberrations through mitosis in human lymphocytes

Stable and unstable chromosome aberrations in human lymphocytes exposed to 2 and 4Gy of X-rays in G(0) were analyzed in M1 and M2 cells harvested at 72h to investigate how the scoring protocol influences the yields of aberrations transmitted through one mitosis. Metaphase chromosomes 2, 3, and 5 were painted using fluorescence in situ hybridization (FISH) whole chromosome probes, together with a pan-centromeric probe and stained by the harlequin-FISH method, to allow the cell cycle status of each cell to be determined as it was scored. A strict scoring criterion was adopted so that each metaphase had to contain 46 centromeres and each dicentric/centric ring had to have an acentric present. In addition to scoring the painted material, unstable aberrations in the whole genome were also recorded. The yield of complete dicentrics decreased by more than a factor of 2 in going from M1 to M2. The decrease was greater at the lower dose. Two-way translocations appeared stable, but one-way translocations decreased. This suggests that if translocation yields are to be used for biological dosimetry purposes, then the two-way type should be used.     

Differential sensitivity to x-ray of chromosomes of blood T-lymphocytes and B-and T-cell lines.

Normal T-lymphocytes, B-cell line (CCRF-SB) and T-cell line (CCRF-HSB-2) cells, all diploid in their chromosome constitution, were exposed in vitro to various doses of X-ray and analyzed at their first mitotic division for structural chromosome abnormalities. The irradiation effects were determined also by a viability test of the cells, using trypan blue dye. The irradiated T-cell line (CCRF-HSB-2) showed a remarkably high frequency of chromosome aberrations, including chromosome and chromatid deletions, chromatid exchanges, dicentrics, rings and acentric fragments. On the other hand, the chromosome aberrations observed in the irradiated B-cell line and normal T-lymphocytes consisted mainly of dicentrics, rings, deletions and acentric fragments; the frequency of chromosome and chromatid deletions was low as compared to that of the T-cell line. The cell viability test showed a singificantly higher percent reduction of viable cells at every dose of X-ray in the irradiated T-cell line than in the B-cell line or the normal T-lymphocytes. It is possible that the increased radiosensitivity of the T-cell line is related to the original malignant nature of the cells, which originated from the lymphocytes of a patient with acute lymphoblastic leukemia.     

Detection of chromosome aberrations by FISH as a function of cell division cycle (harlequin-FISH)

Chromosome aberrations are a sensitive indicator of genetic change, and the measurement of chromosome aberration frequency in peripheral blood lymphocytes is often used as a biological dosimeter of exposure (1,4). The length of time that cells are maintained in culture before cytogenetic analysis is probably the most important in vitro factor that influences both the frequency and types of aberrations that are seen following exposure to mutagens. Therefore, for accurate cytogenetic measurements of genetic damage, cells must be analyzed in their first mitosis following exposure. As cells progress through subsequent mitotic division cycles, cells with unstable types of aberrations, e.g., dicentrics and acentric fragments, are eliminated (1,3,4). Even the use of synchronized populations of cells does not guarantee that all cells analyzed will be in their first division following treatment. Small variations in growth rate after irradiation can lead to large variations in the proportion of cells that are in their first vs. a subsequent mitosis. For example, 48 h after G0 lymphocytes are stimulated to enter the cell cycle (the standard sampling time for cytogenetic analysis), up to 50% of the cells in mitosis can be in their second division cycle (10). While there are methods available to distinguish cells in different division cycles (see Introduction), they are not easily adapted for use with standard fluorescence in situ hybridization (FISH) procedures. The goal of this study was to develop a simple approach to detect aberrations by FISH whereby cells in different division cycles could be distinguished.     

Differential sensitivity to X-ray of chromosomes of blood T-lymphocytes and B- and T-cell lines

Summary Normal T-lymphocytes, B-cell line (CCRF-SB) and T-cell line (CCRF-HSB-2) cells, all diploid in their chromosome constitution, were exposed in vitro to various doses of X-ray and analyzed at their first mitotic division for structural chromosome abnormalities. The irradiation effects were determined also by a viability test of the cells, using trypan blue dye. The irradiated T-cell line (CCRF-HSB-2) showed a remarkably high frequency of chromosome aberrations, including chromosome and chromatid deletions, chromatid exchanges, dicentrics, rings and acentric fragments. On the other hand, the chromosome aberrations observed in the irradiated B-cell line and normal T-lymphocytes consisted mainly of dicentrics, rings, deletions and acentric fragments; the frequency of chromosome and chromatid deletions was low as compared to that of the T-cell line. The cell viability test showed a significantly higher percent reduction of viable cells at every dose of X-ray in the irradiated T-cell line than in the B-cell line or the normal T-lymphocytes. It is possible that the increased radiosensitivity of the T-cell line is related to the original malignant nature of the cells, which originated from the lymphocytes of a patient with acute lymphoblastic leukemia. Supported in part by USPHS grants CA-14413 and CA-16935.     

Comparison of cytogenic response of human lymphocytes to in vivo and in vitro exposure to low doses of gamma-rays. Unstable chromosomal exchanges detected by FPG techniques

On peripheral lymphocytes of eight cancer patients undergone whole-body therapeutic irradiation (at daily dose of 10 cGy up to total dose of 50 cGy of 60Co gamma-rays) the dose-response of unstable chromosome exchanges (dicentrics and centric rings) was studied. This dose response fitted well linear function. The lower slope of dose-response curve was found for in vivo irradiated lymphocytes as compared to the dose response curve obtained for in vitro irradiated lymphocytes of the same patients. This finding seems to provide evidence that in case of protracted irradiation of individuals an absorbed dose could be underestimated if for biological dosimetry an in vitro dose response curve for unstable chromosome aberrations is used as referent one.     

Chromosome aberrations induced in human lymphocytes by 3.45 MeV alpha particles analyzed by premature chromosome condensation.

Premature chromosome condensation (PCC) experiments using human lymphocytes with centromere staining have shown that after exposure to 3.45 MeV alpha-particle radiation, the full number of dicentric chromosomes appears when the cell fusion protocol is applied immediately after irradiation. In this case, the time available for repair and misrepair of DNA damage is only about 30 min. The number of dicentrics does not change with a further increase in the time available for chromatin rearrangement. This fast response confirms the expectation based on our previous experiments using PCC with 150 kV X rays in which the alpha component of the yield of dicentrics was found to appear when the cell fusion protocol was applied immediately after irradiation, whereas the beta component was delayed by several hours. The time constant for rejoining of the excess acentric chromosome fragments is found to be donor-specific and not to differ for alpha particles and X rays, but alpha-particle radiation leaves a larger fraction of the excess acentric fragments unrejoined. The RBEs of the 3.45 MeV alpha-particle radiation compared to 150 kV X rays, evaluated for the alpha component for the yield of dicentrics and for the yield of unrepaired acentric fragments, have almost equal values of about 4. This is consistent with data in the literature on chromosome aberrations observed in metaphase that show the equality of the RBE values for production of dicentrics and acentric fragments. Our experimental results concerning the fast kinetics of the alpha component of the yield of exchange-type chromosome aberrations are not consistent with Lea's pairwise lesion interaction model, and they support the proposed alternative mechanism of lesion-nonlesion interaction between chromatin regions carrying clustered DNA damage and intact chromatin regions.     

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).     

Results of dynamic cytogenetic study of children and teenagers living in areas, radioactive by contaminated after the Chernobyl accident

Results of multiyear cytogenetic study of children and teenagers living in areas, radioactive by contaminated after Chernobyl accident, were adduced. Mean density of 137Cs contamination in two compared living areas were 111 and 200 kBq/m2 and mean external doses accumulated for 1986-2001 were 6.7 and 11.4 mGy correspondingly. Averaged thyroid doses receives by subjects of all age groups in the second area were approximately 1.5 times higher than in the first area; in the youngest group (0-1 year) the doses were 114.3 and 174.3 mGy. During 17 years cytogenetic investigation approximately from 30% to 60% of examined persons were observed the increased level of chromosome aberrations in lymphocytes of peripheral blood. Average frequency of unstable aberrations (acentrics, dicentrics and centric rings) constituted about 0.4 per 100 cells (0.22 per 100 cells in controls) during all period of observation. Level of marker aberrations (dicentrics and centric rings) was increased almost all times of study and varied within 0.04-0.19 per 100 cell (0.03 in control group). The parallel investigation of frequency of stable aberrations by FISH method showed up their level about 3 times exceeding observed dicentrics level. Comparably higher indexes of cytogenetic disturbances were revealed in group exposed in utero during period of accident.     

Some problems of spontaneous and induced mutagenesis in mammals and man.

The culture time of rabbit lymphocytes (41–42 h) that provides cells in their first post-stimulation mitosis, was estimated on the basis of the mitotic index, dicentric yield and presence of the cells with these aberrations unaccompanied by acentric fragments, studied as a function of culture duration. The cells obtained in metaphase from cultures terminated at this time displayed no donor-to-donor variation where induction of dicentrics by X-rays was concerned.Rabbit venous blood was irradiated in vitro with a range of X- and gamma-ray doses, and dose-effect curves were obtained by regression analysis. Sixteen rabbits were irradiated in vivo (uniform whole-body irradiation), and blood was sampled 10 min, 6, 24, and 48 h after exposure. The frequency of dicentrics in the lymphocytes cultured did not change significantly over the first 24 h after irradiation. Dose-effect relationships in vivo fell within one standard error confidence limits of the respective curves in vitro. The authors conclude that the latter may be used for estimation of dose in vivo under conditions of homogeneous whole-body irradiation.     

The induction of chromosome aberrations during the course of radiation therapy for morbus Hodgkin

In a patient with Morbus Hodgkin, structural aberrations of the chromosome type in peripheral lymphocytes were analyzed during radiation therapy (accumulated target dose 44.6 Gy: 22 fractions of 1.8 Gy each and 2 fractions of 2.5 Gy each at the end of the therapy). The blood was sampled about 5 min after a fraction and/or 24, 48, or 72 h thereafter. The frequency of dicentric chromosomes:acentric fragments:centric ring chromosomes is 37:14:1 throughout the therapy. Independent of the time of blood sampling after a fraction, the distributions of dicentrics and acentrics are overdisperse and represent negative binomial distributions. The yields from these aberrations, as determined during the course of radiotherapy, are best fitted to a linear-quadratic function with a negative quadratic term. The two dose-effect curves (blood sampling about 5 min and 24 to 72 h after a fraction) of dicentrics and acentrics do not differ significantly. Up to an accumulated target dose of about 20 Gy the percentages of cells with chromosome aberrations increase to about 48 to 65% and, at this level, remain constant until the end of therapy.     

Long-term follow-up cytogenetic survey and biological dosimetry in persons evacuated from 30-km Chernobyl NPP zone

The paper presents the results of the follow-up cytogenetic survey and biological dosimetry carried out in inhabitants of Pripiat' town and nearby villages, who were departured from the Chernobyl NPP 30-km exclusive zone during first days after the Chernobyl catastrophe. The unstable chromosome aberration level in inhabitants were significantly increased above control in terms up to 1 year after evacuation and declined gardually during next 14 years. In early period the cytogenetic damage frequency in evacuees showed no dependence on gender. The chromosome type aberration level appeared to be lower in young persons comparing with adults. The dicentrics plus centric rings yield had a positive correlation with duration of staying at Chernobyl zone. The average doses of protracted exposure were calculated from the dicentrics and centric rings yields; the dose estimations appeared to be 1.4 times higher in persons evacuated 3-11 days after the accident than that of in persons with shorter departure time. Uing the Bayesian analysis the probabilistic distribution of biological doses was constructed for the studied evacuees group. This distribution was characterized by a mean dose of 360 mGy, the modal doses of 200-450 mGy and 80% of probability density within the dose range 0-1000 mGy, that seems to be sufficient for considering the increased risk of late somatic radiation effects for this cohort.     

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.     

Retrospective dose estimation in remote period after exposure using different biological methods

Investigation of application of chromosome aberrations of lymphocytes in peripheral blood for biological dosimetry purposes in remote (up to 40 years) period after acute exposure to doses of 1 Gy and more was carried out. The comparative analysis of frequency of unstable and stable (using FISH and G-banding methods) aberrations was performed for 24 subjects accidentally exposed to radiation on nuclear submarines during 1961-1985. Statistically significant increasing of frequency of dicentrics and centric rings was determined in the exposed subjects in remote period after exposure to compare with controls. Their sum frequency in the exposed group varied depending on ARS heaviness from 0.1 to 1.0 aberrations per 100 cells. In control group it was from 0 to 0.2 correspondingly. Translocation frequency (complete + incomplete) fixed by FISH method (2, 4, and 12 chromosomes) varied within the limits of 0.2-16.0 for exposed subjects and 0.3-1.26 translocations per genome per 100 cells for controls. Some examined persons (5 subjects) exposed to accident in 1985 had results of analysis of unstable chromosome aberration in acute period after exposure that allow to estimate obtained doses by dicentrics frequency which having good correlation with ARS heaviness. Individual dosed using traslocation frequency were defined retrospectively in 11 from 21 exposed persons. They correlate with calculated physics doses and doses estimated by haematolotical parameters in acute period and also doses obtained by ESR spectroscopy of tooth enamel in remote period.     

Persistence of chromatid aberrations in the cells of solid mouse tissues exposed to 137Cs gamma radiation

Primary mouse ear and kidney cultures were established for determination of cytogenetic aberrations at short (3 days to 1 month) and long (12-23 months) times after exposure of their right sides to 7.5 Gy of (137)Cs gamma radiation. In every case, higher levels of aberrations were observed in primary cultures established from the irradiated tissues than in those established from the contralateral tissues. The most common aberrations in the contralateral tissues and those from nonirradiated mice were chromatid and isochromatid breaks and small chromatid fragments. Primary cells from irradiated tissues removed from animals within a month of exposure displayed a variety of unstable chromosome-type aberrations characteristic of recent exposure to ionizing radiation including rings, dicentrics, double minutes, and large acentric fragments. The percentages of cells exhibiting chromatid breaks and small chromatid fragments were also markedly elevated. Although the levels of chromosome-type aberrations found in primary cells from irradiated tissues dropped to near background levels a year or more after exposure, chromatid-type aberrations remained elevated. These results are consistent with long-term persistence of damage in the genomes of ionizing radiation-exposed cells in solid tissues and the induction of genomic instability in vivo.     

The ratio of dicentrics to centric rings produced in human lymphocytes by acute low-LET radiation.

Chromosome aberrations produced by ionizing radiation are assumed to develop from DNA double-strand breaks (DSBs) which interact pairwise, in an exchange event. Dicentrics and centric rings are aberrations that exemplify inter- and intrachromosomal exchanges, respectively. We show from a survey of published data that for acute low-LET irradiation of resting human lymphocytes the observed ratio of dicentrics to centric rings is approximately five times smaller than predicted by a pairwise interaction model which assumes complete randomness. Such a low ratio can be interpreted as evidence for a proximity effect, favoring exchanges of an intrachromosomal type. That is, since DSBs induced close together have an above-average chance of pairwise interaction, the observed excess of centric rings indicates that at the time of irradiation there is some degree of spatial confinement for the two arms of a single chromosome. Assuming the excess of centric rings is indeed due to proximity effects, the data are used to estimate that the volume of a domain, within which any one lymphocyte chromosome is localized at one instant during the G0/G1 phase, is at most approximately 20% of the nuclear volume.