Chromosome aberrations and radiation-induced cell death. I. Transmission and survival parameters of aberrations

Synchronous cultures of V₇₉ Chinese hamster cells were irradiated in G₁ with 300 rad of X-rays. Cells were collected for 2-h intervals after synchronization to include the first three post-irradiation divisions and were scored for chromosome aberrations. After the first post-irradiation division, asymmetrical exchanges were distributed according to the Poisson formula and both the asymmetrical exchange frequency and the acentric fragment frequency exhibited significant variations with collection time. Formulae derived from a previous mathematical analysis were used in conjunction with the aberration frequencies observed at the first, second, and third post-irradiation divisions to predict transmission and survival parameters for specific chromosomal aberrations.The probability, 2T, that an acentric fragment will be transmitted to a daughter cell at anaphase was found to be 0.57. The probability, W, that a two-break aberration (asymmetrical exchange) will be transmitted and observed at the next division was 0.56. Finally, the probability, P, that a cell will survive to a subsequent mitosis after losing a single acentric fragment was about 1.0 for one post-irradiation generation but somewhat less for two generations.     

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.     

Transmission of radiation-induced acentric chromosomal fragments to micronuclei in normal human fibroblasts

A simplifying assumption made when calculating the probability of a chromosomal aberration resulting in a micronucleus is that virtually all radiation-induced micronuclei result from acentric fragments. In the present study we used antibodies to chromosomal centromeres (kinetochores) to determine the frequency of centric versus acentric micronuclei in normal human fibroblasts exposed to 6 Gy of 60Co gamma rays while they were in density-inhibited growth. Up to 14% of the micronuclei induced by this exposure contained one or more kinetochores; i.e., they were not composed of acentric chromatin. By deleting kinetochore-positive micronuclei from the analysis, and by reconstructing micronucleus frequencies based on the fraction of cells that had divided following radiation exposure, a direct comparison between micronuclei and acentric chromosome fragments was made. On that basis, the probability of an acentric fragment becoming a visible micronucleus in either daughter cell of a dividing pair was estimated to be about 0.6. The distribution of acentric fragments among mitotic cells conformed to Poisson expectation, while the distribution of micronuclei among daughter cells was significantly overdispersed. The phenomenon of overdispersion is discussed in connection with proposed cellular processes that effect a nonrandom segregation of acentric fragments.     

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.     

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.     

Chromosomal aberrations and sister-chromatid exchanges in tool and die workers

Because tool and die workers are exposed to a number of potentially genotoxic agents, including mutagenic metals, polyaromatic hydrocarbons, and nitrosamines, and may be at increased cancer risk, the present study was undertaken to test whether chromosomal damage in peripheral blood cells is associated with work in the tool and die industry. Lymphocyte cultures were established from 27 tool and die fabrication workers from one manufacturing plant who had worked in the trade for more than 15 years. 15 of these workers also had some form of malignancy at the time of the study, but had not been treated with radiation or chemotherapies that could themselves induce chromosomal damage. The frequencies of chromosomal aberrations and sister-chromatid exchanges (SCEs) were measured in workers and the data compared with those of a control group consisting of 7 non-fabrication workers from the same plant and 8 age-matched community controls. In addition, the relative rates of lymphocyte proliferation were estimated for each group by analyzing the percentages of first-, second- and third-division mitotic cells after 72 h of culture. The results of the chromosomal studies show that tool and die workers have significantly increased frequencies of aberrations whether engaged in fabrication work or not, compared to control subjects. The frequency of SCEs and the frequencies of 1st, 2nd and 3rd division figures are not different among the study groups. Among workers who are engaged in fabrication, including those who are cancer patients, the frequency of more complex aberrations (i.e., interstitial deletions and small acentric fragments) is increased. In a five-year follow-up of these workers, 2 of the 13 workers with these aberrations developed some form of colon cancer. Whether the presence of interstitial deletions and small acentric fragments is related to the occupation of these workers, or is tangentially related to the development of cancer, is presently under consideration.     

Molecular biology of double-minute chromosomes

Double-minute chromosomes play a critical role in tumor cell genetics where they are frequently associated with the overexpression of oncogene products. They have been observed for many years in light microscopic examinations of metaphase chromosomes from tumor cells, but their origin remains unknown and is the subject of considerable speculation. However, molecular details of their structure and organization can now be described in conjunction with the microscopic examinations, to allow an evaluation of the various models that have been developed to explain the genesis of double-minutes. The evidence now favors simple models that invoke chromosome breakage and circularization of very large acentric chromosome fragments, permitting unequal segregation of the genes on the fragment during cell division. If there is selection for overexpression of one of the genes on the fragment, daughter cells with more fragments will grow faster than daughter cells with fewer fragments, and over time the population of cells will come to contain many double-minutes per cell.     

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.     

A branching process model of gene amplification following chromosome breakage.

We have devised a mathematical model of gene amplification utilizing recent experimental observations concerning dihydrofolate reductase (DHFR) gene amplification in CHO cells. The mathematical model, based on a biological model which proposes that acentric elements are the initial intermediates in gene amplification, includes the following features: (1) initiation of amplification by chromosomal breakage to produce an acentric structure; (2) replication of acentric DNA, once per cell cycle; (3) dissociation of replicated acentric DNA; (4) unequal segregation of acentric DNA fragments to daughter cells at mitosis; (5) subsequent reintegration of acentric fragments into chromosomes. These processes are assumed to be independent for each element present in a cell at a given time. Thus, processes of unequal segregation and integration may occur in parallel, not necessarily in a unique sequence, and may be reiterated in one or multiple cell cycles. These events are described mathematically as a Galton-Watson branching process with denumerable infinity of object types. This mathematical model qualitatively and quantitatively reproduces the major elements of the dynamical behavior of DHFR genes observed experimentally. The agreement between the mathematical model and the experimental data lends credence to the biological model proposed by Windle et al. (1991), including the importance of chromosome breakage and subsequent gene deletion resulting from resection of the broken chromosome ends as initial events in gene amplification.     

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.     

Polar ejection forces are operative in crane-fly spermatocytes, but their action is limited to the spindle periphery.

Laser microsurgery was employed to reveal kinetochore-independent forces acting on chromosome arms in crane-fly spermatocytes. When a portion of an arm situated along the interpolar axis between the equator and a pole was cut off, the resultant acentric fragment was transported poleward and outward into the peripheral domain of the spindle. If the fragment was generated well in advance of the onset of anaphase, then at the spindle periphery, it changed direction and moved away from the pole and back toward the equator. That domain-specific movement-poleward in the central spindle and away from the pole at the spindle periphery-not only provides the first evidence for polar ejection forces acting on acentric fragments in a meiotic system, but it is the first example of kinetochore-independent forces in both directions at the same stage of division. Sniglets generated by laser pulses directed at specific sites in the spindle revealed that the mechanism underlying ejection forces was specific to chromosomes. At anaphase onset, polar ejection forces ceased, and pole-directed forces took over. At that time, chromosome fragments that had been ejected to the equator moved poleward again, providing clear evidence for kinetochore-independent forces on chromosome arms during anaphase.     

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.     

Use of the frequencies of micronuclei as quantitative indicators of X-ray-induced chromosomal aberrations in human peripheral blood lymphocytes: comparison of two methods

The yield of radiation-induced micronuclei in human lymphocytes was assessed by two methods, i.e., by incorporating bromodeoxyuridine or by inhibiting cytokinesis by cytochalasin for identification of cells which have undergone one cell division. The cytochalasin block method was found to be more efficient with a capacity to detect between 60 and 90% of the induced fragments. Dose-response characteristics and the results of fractionation experiments indicate that the yield of micronuclei reflects both classes of acentric fragments, i.e., those associated and independent of exchange type of aberrations.     

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.     

Linear dose-response of acentric chromosome fragments down to 1 R of x-rays in grasshopper neuroblasts, a potential mutagen-test system

Grasshopper-embryo neuroblasts have no spontaneous chromosome breakage; therefore they permit easy detection of agents that break chromosomes. An X-ray exposure of 1 R induces in them a detectable number of chromosome fragments. The dose-response of acentric fragment frequency fits a linear model between 0 and 128 R. Thus another cell type is added to those previously demonstrated to have no threshold dose for the induction of chromosome or gene mutations.     

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.     

A comparison between short-term evolution of micronuclei induced by X-rays and colchicine in root tips of Vicia faba

The short-term evolution of micronuclei derived from acentric fragments and whole chromosomes was studied in root tips of Vicia faba. Micronuclei were induced by X-rays (30 cGy and 120 cGy) and colchicine (10(-5) M and 3 X 10(-4) M). Frequencies of chromosome breakage or loss of micronuclei in interphase and mitotic cells were studied. The DNA content of micronuclei in interphase cells was also measured. Micronuclei derived from whole chromosome showed a higher probability to survive and to undergo mitotic condensation in synchrony with main nuclei than micronuclei derived from an acentric fragment. PCC (Premature Chromosome Condensation) was not observed for both types of micronuclei in Vicia faba, in contrast to the ones reported in mammalian cells in culture.     

A rapid procedure for DNA sequencing using transposon-promoted deletions in Escherichia coli

A simple procedure has been developed for sequencing long fragments of DNA. The fragment (which can be several kb in length) is cloned in pAA3.7X, and subdivided into many overlapping segments by Tn9-promoted deletions. The deletions are isolated by positive selection for galactose resistance. A rapid plasmid preparation from several hundred galactose-resistant colonies is fractionated by agarose gel electrophoresis to pick a series of deletions terminating at approx. 200-bp intervals across the entire length of the fragment. Selected plasmids are purified by rapid alkaline extraction, and used directly for supercoil sequencing with a primer derived from IS1. Sequences of adjacent deletions contain overlaps which are used to connect individual sequences to give the complete sequence.     

Unfolding-refolding behaviour of chicken egg white ovomucoid and its correlation with the three domain structure of the protein

The urea and heat-induced unfolding-refolding behaviours of chicken egg white ovomucoid and its four fragments representing domains I, II + III, I + II and III were systematically investigated in 0.06 M sodium phosphate buffer (pH 7.0) by difference spectral measurements. The effect of temperature on ovomucoid and its fragments was also studied in 0.05 M sodium acetate buffer (pH 5.0) and in presence of 2 M urea at pH 7.0. Intrinsic viscosity data showed that ovomucoid and its different fragments did not lose any significant amount of their structure under mild acidic conditions (pH 4.6). Difference spectral results showed extensive disruption of the native structure by urea or temperature. Isothermal transitions showed single-step for domain I, domain I + II and domain III, and two-step having one stable intermediate, for ovomucoid and its fragment representing domain II + III. However, the presence of intermediate was not detected when the transitions were studied with temperature at pH 7.0. Strikingly, the single-step thermal transitions of ovomucoid and its fragment representing domain II + III, became two-step when measured either at pH 5.0 or in presence of 2 M urea at pH 7.0. Analysis of the equilibrium data on urea and heat denaturation showed that the second transition observed with ovomucoid or domain II + III represent the unfolding of domain III. The kinetic results of ovomucoid and its fragments indicate that the protein unfolds with three kinetic phases. A comparison of three rate constants for the unfolding of intact ovomucoid with that of its various fragments revealed that domain I, II and III of the protein correspond to the three kinetic phases having rate constants 0.456, 0.120 and 0.054 min-1, respectively. These data have led us to conclude: (i) the unusual stability of ovomucoid towards various denaturants, including temperature, is due to its domain III, (ii) initiation of the folding of the ovomucoid molecule starts from its NH2-terminal region which probably provides the nucleation site for the formation of the subsequent structure and (iii) domains I and II have greater mutual recognition between them as compared to the recognition either of them have with domain III.     

Radiobiological effects of a low-energy ion beam on wheat

The radiobiological effects of a low-energy nitrogen ion (N⁺) beam on wheat were studied, particularly with regard to the induction of chromosome aberrations. The results demonstrated that the three test varieties showed different sensitivities to ion implantation, and a higher dose of ion implantation had a marked effect on the germination and survival rate of the seeds exposed. The germination rate and survival rate curve basically followed a similar trend in the same variety. Cytological analysis indicated that ion beams were effective in producing chromosome aberrations. The frequencies of mitotic or meiotic cells with chromosome aberrations increased linearly with increasing doses. The aberration types included, for example, acentric fragments, chromosome deletions, lagging chromosomes, chromosome bridges and micronuclei. In the root tip cells, aberrations chiefly consisted of acentric fragments and deletions. Chromosome bridges and lagging chromosomes were the main aberration phenomena observed in the pollen mother cells. The highest frequencies of root tip cells and pollen mother cells with chromosome aberrations were 15.2% and 39.8%, respectively. Changes in morphology and mutant were also observed in the plants derived from exposed seeds. Received: 10 April 2000 / Accepted: 10 October 2000