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.     

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.     

Use of unstable chromosome aberrations for biological dosimetry after the first postirradiation mitosis

The loss of unstable chromosome aberrations after the first postirradiation mitosis makes their use difficult in radiation dosimetry. We describe here a method which, in a cell population observed at this stage, allows retrospective estimation of the frequencies of the unstable aberrations induced at the time of irradiation, and their use as a dosimeter. The laws controlling the behavior of unstable aberrations during mitosis were defined from a large-scale experiment on irradiated human lymphocytes. For cells undergoing the first, second, or third mitosis after irradiation, relationships were determined between the frequency, at irradiation time, of acentric fragments not arising from formation of dicentrics or rings, and the ratio of dicentrics and centric rings appearing without acentric fragments to the total number of dicentrics plus rings. On the basis of this ratio, the method described here provides an assessment of the postirradiation mitotic activity in a cell population. This assessment permitted estimation of the cell distribution and frequency of dicentrics plus centric rings, and of the frequency of acentric fragments at the time of irradiation. The use of this method for retrospective dosimetry after whole-body irradiation under various conditions of exposure is illustrated.     

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.     

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.     

Enhanced expression of X-ray- and UV-induced chromosome aberrations by cytosine arabinoside in ataxia telangiectasia cells

The effect of cytosine arabinoside (ara-C) on the frequency of X-ray- or UV-induced chromosome aberrations was studied in cultured skin fibroblasts derived from 2 normal persons, 4 ataxia telangiectasia (AT) patients and 2 obligate AT heterozygotes. Density-inhibited cells were irradiated with X-rays or UV, post-treated with ara-C, and chromosomes in the first post-irradiation mitoses were examined. UV, a poor inducer of chromosome-type aberrations in G1, caused chromosome-type aberrations (dicentrics and rings) when coupled with ara-C both in normal and AT cells, but to a much greater extent in AT cells. In AT cells, an elevated induction of both terminal deletions and chromatid aberrations was also observed by the application of UV and ara-C, and unexpectedly, UV alone induced a considerable frequency of both types of aberrations. The enhancing effect of ara-C on X-irradiated cells was less pronounced than on UV-irradiated cells. The responses of AT heterozygotes were virtually the same as those of normal cells. These findings suggest that ara-C can convert the UV-induced DNA damage into the type that has a potential to induce dicentrics and rings in G1 as well as to elicit a hypersensitive response of AT cells.     

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).     

Increase in the frequency of gamma-ray induced chromosomal aberrations in mammalian cells by post-treatment with novobiocin

The effect of novobiocin (an inhibitor of DNA topoisomerase and polymerase) on the frequency of chromosomal aberrations was examined in Chinese hamster V79 cells irradiated with gamma-rays in the plateau phase of growth and subcultured in the presence of novobiocin until the first mitosis after irradiation. Novobiocin alone affected cell survival, DNA synthesis and the mitotic frequency of unirradiated cells in a dose-dependent manner, without causing any significant increase in the frequency of chromosome- or chromatid-type aberrations. The frequency of chromosome-type aberrations induced by gamma-radiation was not influenced by novobiocin at 200 microM, but the frequency of chromosome deletions (but not rings and dicentrics) showed a two-fold increase when 300 microM novobiocin was present. Irradiation produced a low level of chromatid-type aberrations and post-treatment with novobiocin at concentrations greater than 100 microM significantly increased the frequency of chromatid gaps and breaks. The results support the idea that different radiation-induced lesions lead to chromosome- as opposed to chromatid-type aberrations.     

The biological activity of hydrogen peroxide. I. Induction of chromosome-type aberrations susceptible to inhibition by scavengers of hydroxyl radicals in human embryonic fibroblasts

The cytogenetic effect of hydrogen peroxide (H2O2) was investigated in human embryonic fibroblasts. Chromosome-type aberrations were found together with chromatid-type aberrations in metaphase cells harvested 24 h after a single 10-min treatment with 10(-5)-10(-3) M H2O2 in 0.9% NaCl solution. The chromosome-type aberrations were observed to be predominantly dicentrics and deletions. Both types of aberration showed a dose-response relationship to the dose of H2O2 over the range of 10(-5)-1.5 X 10(-4) M H2O2. The intercellular distribution of dicentrics showed a Poisson distribution. Centric and acentric rings and abnormal monocentrics were a minor fraction of the chromosome-type aberrations. The chromatid-type aberrations observed, such as breaks, exchanges and gaps, showed no dose-response relationship. The frequency of isochromatid breaks was higher than that of chromatid breaks and approximately 70% of the isochromatid breaks were found in the centromeric or pericentromeric region. The intercellular distribution of chromatid exchanges showed an over-dispersed distribution. The generation of aberrations by H2O2 was effectively suppressed by catalase and several scavengers of hydroxyl radicals (.OH) such as ethanol, dimethyl sulfoxide (DMSO) and mannitol. This result suggest that .OH plays an essential role in the generation of the chromosome aberrations by H2O2.     

Analysis of the Chromosome Aberrations Induced by X-Rays in Somatic Cells of DROSOPHILA MELANOGASTER

A technique has been perfected for enabling good microscope preparations to be obtained from the larval ganglia of Drosophila melanogaster. This system was then tested with X-rays and an extensive series of data was obtained on the chromosome aberrations induced in the various stages of the cell cycle.-The analysis of the results obtained offers the following points of interest: (1) There exists a difference in radio-sensitivity between the two sexes. The females constantly display a greater frequency of both chromosome and chromatid aberrations. They also display a greater frequency of spontaneous aberrations. (2) In both sexes the overall chromosome damage is greater in cells irradiated in stages G(2) and G(1). These two peaks of greater radiosensitivity are produced by a high frequency of terminal deletions and chromatid exchanges and by a high frequency of dicentrics, respectively. (3) The aberrations are not distributed at random among the various chromosomes. On the average, the Y chromosome is found to be more resistant and the breaks are preferentially localized in the pericentromeric heterochromatin of the X chromosome and of the autosomes. (4) Somatic pairing influences the frequency and type of the chromosome aberrations induced. In this system, such an arrangement of the chromosomes results in a high frequency of exchanges and dicentrics between homologous chromosomes and a low frequency of scorable translocations. Moreover, somatic pairing, probably by preventing the formation of looped regions in the interphase chromosomes, results in the almost total absence of intrachanges at both chromosome and chromatid level.     

Mapping by transduction of mutator gene mutH in Escherichia coliz

Spontaneous levels of structural chromosomal aberrations in human peripheral lymphocytes were studied cytogenetically in 49 female and 56 male subjects. With a total of 16 267 metaphase spreads examined, 191 cells were found to contain chromosomal aberrations, giving a rate of 1.17%. The rates of individual aberration types were as follows: chromatid fragments, 0.39%; chromosome fragments, 0.71%; dicentrics, 0.06%; symmtrical and asymmetrical chromatid exchanges, 0.01%; and rings, 0.006%. There were significant differences in aberration yields between females aged 20–29 yr. and 60–70 yr., whereas males aged 20–50 yr. showed no difference. The two sexes differed significantly in chromatid fragments, chromosome fragments and aberrant cells.     

The role of cytogenetic examination for prognosis of remote consequences of irradiation

Correlation between the level of somatic pathology and cytogenetic characteristics of blood was analyzed in a group of liquidators of the accident at the Chernobyl Nuclear Power Plant (ChNPP). A statistically significant correlation was found between the occurrence of cardiovascular diseases and the level of chromosome aberrations (total frequency of chromosome aberrations, frequency of dicentrics and centric rings, frequency of chromatid aberrations). The results obtained are of great importance for the substantiated prediction of the development of postradiation pathologies.     

The fate of X-ray-induced chromosome aberrations in blood lymphocyte culture

The BrdU-Giemsa method which facilitates an unequivocal identification of metaphases at different cycles has been utilized to investigate the fate of X-ray-induced chromosome aberrations in the blood lymphocyte culture system of the Indian muntjac which has the lowest diploid number (2n = 6 female/7 male) and easily distinguishable large-sized chromosomes. The results demonstrate that about 50% of dicentrics and only 12% of rings were transmitted from the first cycle to the second. There were as high as 73% abnormal cells in the second cycle as against 94% in the first cycle following 4.0 Gy. However, the frequencies of dicentrics, rings and of abnormal cells were greatly reduced in the third+ cycles. The frequencies of acentric fragments per post-irradiated first, second and third+ division cell were 2.21, 0.64 and 0.24, respectively. In sharp contrast to all earlier reports, about 75% of them were retained as a single acentric fragment in the second cycle. Analysis of fragment segregation during anaphase separation supports this finding. The survival probability of dicentrics and rings was found to be more than 60% in the second and only 18% in the third+ cycle.     

Cytogenetic analysis of children under long-term antibacterial therapy with nitroheterocyclic compound furagin

Cytogenetic analysis of chromosome aberrations (CAs) and sister chromatid exchanges (SCEs) was performed in 109 blood samples from 95 pediatric patients with urinary tract infections (UTIs). Children were exposed to diagnostic levels of X-rays during voiding cystourethrography and subsequently treated for one to 12 months with low doses of furagin - N-(5-nitro-2-furyl)-allylidene-1-aminohydantoin. Furagin is 2-substituted 5-nitrofuran, chemically and structurally similar to well-known antibacterial compound nitrofurantoin. Increased frequencies of CAs were found in children undergoing voiding cystourethrography as compared with the unexposed, acentric fragments being the most frequent alteration (2.03 versus 0.88 per 100 cells, P=0.006). However, a significant decrease in the frequency of acentric fragments was determined with the time elapsed since X-ray examination was performed. A time-independent increase in SCE frequency was found in lymphocytes of children treated with furagin. Total CA frequency did not differ significantly between groups of children with various duration of furagin treatment. However, frequency of chromatid exchanges (triradials and quadriradials) increased significantly with duration of treatment.     

A Formula to Predict the Transmission Frequency of Acentric Fragments

A formula, based on the Poisson distribution of radiation-induced chromosomal deletions, was derived to predict the frequency of transmission of acentric fragments between subsequent mitoses. The frequency of deletions observed in the i(th) + 1 division subsequent to fragment distribution at the i(th) division anaphase is independent of the cell death resulting from fragment loss. Further, the transmission frequency of chromosome acentric fragments is mathematically equal to the fragment frequency observed in the i(th) + 1 generation divided by the mean fragment frequency in the i(th) generation. The formula was also extended to chromatid deletions.     

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.     

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.     

Analysis of streptozotocin-induced incomplete chromosome elements and excess acentric fragments in Chinese hamster cells using a telomeric PNA probe

Fluorescence in situ hybridization (FISH) with a telomeric peptide nucleic acid (PNA) probe was employed to analyze the induction of incomplete chromosome elements (ICE, i.e., unjoined or “open” chromosome elements with telomeric signal at only one end) and excess acentric fragments (i.e., in excess of fragments resulting from the formation of dicentric and ring chromosomes) by the methylating agent streptozotocin (STZ) in a Chinese hamster embryo (CHE) cell line. CHE cells were treated with 0–4 mM STZ and chromosomal aberrations were analyzed in the first mitosis after treatment using the telomeric probe. Centric (incomplete chromosomes) and acentric (terminal fragments) ICE were the only unstable chromosome-type aberrations induced by STZ in CHE cells. The induction of these aberrations exhibited a curvilinear concentration–response relationship. About 40% of the metaphases present in cell cultures treated with STZ contained one or more pairs of ICE. In STZ-treated cells, ICE were always observed as pairs consisting of an incomplete chromosome and a terminal fragment. Moreover, all of the excess acentric fragments induced by STZ were of terminal type. These results indicate that chromosomal incompleteness is a very common event following exposure to STZ and suggest that all of the excess acentric fragments induced by STZ originate from terminal deletions.     

Quantitative and qualitative analysis of the effects of various doses of beta-irradiation (50-5000 rads) on epidermal cells of embryos and larvae of Pleurodeles waltlii Mich. (Amphibia, Urodela)

Embryos of Pleurodeles waltlii at the hatching stage were irradiated with doses of 50 to 5 000 rad. From 70 to 500 rad chromosomal aberrations appear; they are studied respectively 24,48 hours and 3 weeks after the treatment. Breakages are observed, that may be followed by rearrangements, i.e. acentric, telocentric and dicentric fragments, chromatid translocations and chromosome translocations. With time, the cells showing the most severe abnormalities are eliminated by the developing larvae. From 1 000 rad cytoplasmic structures (membrane systems and mitochondria) are alterated .     

Chromosomal aberrations in peripheral lymphocytes from healthy subjects as detected in first cell division

Baseline frequencies of chromosomal aberrations were analysed in human peripheral lymphocytes and the influence of age, sex and smoking habits was considered. From 53 healthy subjects (29 males, 24 females) 54,689 exclusively first division cells (M1) were scored. The frequencies of chromosome aberrations per 1000 cells were 1.15 +/- 0.15 dicentrics (dic), 2.6 +/- 0.3 excess acentric fragments (ace) and 7.0 +/- 0.6 chromatid breaks (crb). An age dependency could only be established for ace. Between males and females no differences in any of the aberration types were observed. For heavy smokers (> 30 cigarettes per day) a significant increase was only found for dic (2.5 +/- 0.6 per 1000 cells). Dicentric frequency was compared with background levels of other studies in which results were reported also from exclusively M1 cells. Despite cell cycle control, differences between laboratories can be observed which may be partly influenced by environmental conditions. But on the other hand the mean frequency of dic (excluding heavy smokers) of 0.95 per 1000 cells reported here is consistent for more than one decade. Since such a consistency of the mean frequency of dic is reported also from another laboratory, the conclusion is drawn that especially for the detection of low-level exposures, each laboratory should establish its own base line data, otherwise, the interpretation of the findings is dependent on the selected background level from the literature.