Chromosome aberrations and radiation-induced cell death. II. Predicted and observed cell survival

The surviving fraction of synchronous V₇₉ Chinese hamster cells was measured in two post-irradiation generations after a 300-rad X-ray dose in G₁ by comparing the colony multiplicity in irradiated and control cultures. In addition, the ability of the irradiated population to form colonies was measured immediately after G₁ irradiation or at 6, 32, 75 or 96 h after X-irradiation. Formulae were used in conjunctionwith previously observed transmission and survival parameters of chromosome aberrations² to predict the amount of cell death at any given time after irradiation. The results indicate that the survival pattern of these cells can indeed be predicted on the basis of cell loss from chromosome aberrations.It is likely that an asymmetrical chromosome exchange (dicentric, centric ring, or tricentric) and a chromosome deletion are equally capable of causing cell death, whereas translocations or inversions apparently do not lead to inviability. Furthermore, cell death is rapid: 45% of the total observed death occurs in the first two post-irradiation generations. The initial decrease in viability is caused predominantly by the formation of anaphase bridges, while cell death from fragment loss becomes increasingly important in later generations. In fact, it is probable that, on the average, a cell that loses a single acentric fragment will survive one generation.     

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.     

Measurements of metaphase and interphase chromosome aberrations transmitted through early cell replication rounds in human lymphocytes exposed to low-LET protons and high-LET 12C ions

Inheritable chromosome aberrations (CA) are of concern because cytogenetic damage may trigger the carcinogenic process. Moreover, stability of radiation-induced CA is a prerequisite for meaningful biological dosimetry. CA inheritability arguably depends on the aberration structure, with symmetrical exchanges being favoured over asymmetrical rearrangements, but it is also affected by radiation quality. CA induced by low-LET protons and high-LET 12C ions in G0 peripheral blood lymphocytes were measured in first- , second- and third-generation by combined FISH/harlequin staining of metaphase as well as prematurely condensed interphase chromosomes 1 and 2. As expected, the frequency of non-transmissible (NT) aberrations declined through replication rounds. A radiation-induced arrest occurred prior to first post-irradiation mitosis that prevalently affected aberrant cells. Aberrant cells incurred cycle delays also at subsequent cycles following proton-irradiation but not 12C ion-irradiation. As expected, the frequency of reciprocal translocations remained fairly stable while that of dicentrics was halved at each mitotic round. A significant fraction of complex-type exchanges was found in third-generation cells following both irradiations and appeared to be transmitted relatively more efficiently after protons than 12C ions. A low but stably transmitted frequency of transmissible (T)-type insertions were detected after 12C ions but not after low LET-irradiation. Our data support a differential ability by aberrant cells to progress through post-irradiation mitoses that is influenced by the aberration burden and radiation quality.     

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.     

The fate of chromosome aberrations

Chromosomal aberrations rapidly disappear from populations of dividing cells, but little is known about the details of the process. One may ask, for example, whether a cell with an acentric fragment is virtually certain to die after the first mitosis or whether it has a high probability of surviving to the second. Some recent data on aberrations in cultured human lymphocytes lead to estimates that fragments (and presumably the cells containing them) survive to the next mitosis about 30% of the time and dicentric chromosomes about 50%. These estimates were made without regard for the proliferation of normal cells, however, and so must be somewhat in error. In fact, when cell proliferation is taken into account, the most likely value of survival of the fragment itself is about 80% (when both daughter cells are considered). Probable ranges of this value and of the other parameters considered are presented. It is hoped that this explicit formulation of a mathematical model will encourage further experimental examination of the effect of various aberrations upon cell populations.     

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.     

Molecular mechanisms of the origin of chromosome aberrations and the structural organisation of eukaryotic DNA

Summary A new hypothesis on the appearance of exchange chromosomal aberrations has been suggested. According to this hypothesis, temporal duplex polynucleotide structure should arise during G₁ and G₂ phases during the correction of DNA. The size of the duplex, as a rule, should be restricted to the size of complementary nucleotide sequences in the regions of repetitions. Any polynucleotide break in a duplex zone would result in chromosome breakage and if complementary broken ends interact with each other, then exchange chromosome aberrations may be formed. This hypothesis would explain such previously obscure phenomena as extremely high frequencies of exchanges after mutagen treatment, the nature of mitotic crossing-over, negative interference, change of aberration types before replication, the low frequency of damaged structural genes during aberration formation, etc.     

Cell kinetics, stomatal differentiation, and diurnal rhythm in Allium cepa

Cell kinetics parameters were obtained for the three mitotic divisions leading to formation of stomata in the epidermis of the cotyledon of Allium cepa seedlings. Analysis of mitotic frequencies throughout the course of development showed that the asymmetrical divisions started at about 50 hr after germination, and the symmetrical divisions were first seen a few hours later. Guard mother cell divisions started around 70 hr after germination. The maximal frequency of both symmetrical and asymmetrical division was found between 3 and 5 days after germination, and the highest frequencies of GMC divisions were observed between 6 and 8.5 days. All divisions ceased after 11 days. The three cell populations analyzed displayed diurnal fluctuations of their mitotic frequencies which were characteristic of the type of cell division measured and seemed independent of the region of the cotyledon in which they took place. The symmetrical divisions displayed two diurnal peaks—one at about 0400 and the other at 1600 hr—and the asymmetrical mitoses showed a single peak at about 2200 hr. Atypical asymmetrical divisions were observed in some guard mother cells, suggesting a different developmental sequence for some of the stomatal complexes.     

The induction of chromosome-type aberrations in G1 by methyl methanesulfonate and 4-nitroquinoline-N-oxide,and the non-requirement of an S-phase for their production

Human lymphocyte were treated in G₁ with 4-nitroquinoline-N-oxide (4NQO) and methyl methanesulfonate (MMS) and then incubated in the presence or absence of cytosine arabinoside (ara-C). There was an increase in aberration frequency in those cells incubated with ara-C compared with those treated with 4NQO or MMS alone. This increase was restricted to chromosome-type aberrations. When cells were treated in G₂ with 4NQO and then incubated with ara-C until fixation, there was an increase in deletions compared with cells treated with 4NQO alone. No exchange aberrations were observed following any treatment even when deletion frequencies were high, as in the case with 4NQO plus ara-C treatment. These results suggest that ara-C can inhibit the repair of DNA damage induced by 4NQO and MMS that is converted into aberrations. They also show that the terms “S-dependent” and “S-independent” used to describe the modes of action of chemical clastogens are not valid.     

Elimination of chromosomal aberrations in some varieties of Lathyrus sativus L.

The patterns of elimination of chromosomal aberrations on a quantitative basis with the passage of time in root meristems was investigated in Lathyrus sativus var. P₂₉₃, P₅₈₅ and LC₇₆ following irradiation of dry seeds with 10, 20, 30, 40 and 50 kR γ-rays. The elimination curves for all 3 varieties fitted the equation N₁ = N₀e^?kT. The mode of elimination for each type of aberration was exponential in all the varieties.     

Chromosomal aberrations after induced pluripotent stem cells reprogramming

Induced pluripotent stem cells (iPSCs) are generated from adult cells that have been reprogrammed to pluripotency. However, in vitro cultivation and genetic reprogramming increase genetic instability, which could result in chromosomal abnormalities. Maintenance of genetic stability after reprogramming is required for possible experimental and clinical applications. The aim of this study was to analyze chromosomal alterations by using the G-banding karyotyping method applied to 97 samples from 38 iPSC cell lines generated from peripheral blood or Wharton’s jelly. Samples from patients with long QT syndrome, Jervell and Lange-Nielsen syndrome and amyotrophic lateral sclerosis and from normal individuals revealed the following chromosomal alterations: acentric fragments, chromosomal fusions, premature centromere divisions, double minutes, radial figures, ring chromosomes, polyploidies, inversions and trisomies. An analysis of two samples generated from Wharton’s jelly before and after reprogramming showed that abnormal clones can emerge or be selected and generate an altered lineage. IPSC lines may show clonal and nonclonal chromosomal aberrations in several passages (from P6 to P34), but these aberrations are more common in later passages. Many important chromosomal aberrations were detected, showing that G-banding is very useful for evaluating genetic instability with important repercussions for the application of iPSC lines.     

Analysis of structural and numerical chromosomal aberrations at the first and second mitosis after X irradiation of two-cell mouse embryos

Two-cell mouse embryos were X-irradiated in the late G2 phase in vivo. The first and second postradiation mitoses were analyzed for chromosomal anomalies. The majority of structural aberrations visible at the first mitosis after irradiation were chromatid breaks and chromatid gaps; only a few interchanges and dicentrics were observed. The aberration frequency resulted in a dose-effect relationship which was well described by a linear model. At the second mitosis 29% of the structural aberrations of the first mitosis were counted; the aberration quality changed only slightly. It is discussed whether these aberrations are to be considered "new," "derived," or unchanged transmitted aberrations. Contrary to the results obtained after irradiation of one-cell embryos, little chromosome loss was induced by radiation in two-cell embryos.     

Proceedings: Heterochromatin and chromosome aberrations: a comparative study of normal and tumour cells of mouse with various distributions of heterochromatin.

Seedlings of Crepis capillaris were irradiated after pulse-labelling with tritiated thymidine ([³H]TdR), and both chromosomal aberrations and presence of silver grains were recorded in the same metaphase cells at various intervals throughout the whole mitotic cycle. The following results were obtained: (a) irradiated roots were homogeneous with respect to the number of aberrations, and heterogenous with respect to labelling index (LI); (b) time-effect curves for labelled (L) and unlabelled (U) cells showed no significant difference from one another; (c) no significant quantitative difference of aberration spectra produced in S and G₂ stages was found. These results support the view that the major factor which determines both quantitative and qualitative variation in the production of chromosomal aberrations by radiation is the time lapse between irradiation and fixation rather than relation of the time of irradiation to the time of DNA synthesis. In addition, it was found that labelling with [³H]TdR modifies the effect of radiation on chromosomes.     

Chromosome aberrations in leucocytes of patients with aseptic meningitis

Summary Three patients with aseptic meningitis and two patients with scarlet fever were subjected to chromosome analysis in cultured leucocytes. Information was obtained that leucocytes of the aseptic meningitis patients exhibit random and non-specific chromosome aberrations significantly higher in frequency than the control series from scarlet fever patients. The most common type of the aberration observed was a single chromatid break including a full break in one chromatid. Isochromatid-type breaks, chromatid- and chromosome-type interchanges and acentric fragments were observed less frequently.Contribution No. 641 from the Zoological Institute, Hokkaido University, Sapporo, Japan. This paper was written to dedicate to Professor Dr. Hans Bauer in celebration of his sixtieth birthday, September 27, 1964. Supported by a grant from the Damon Runyon Memorial Fund for Cancer Research to S. Makino, DRG-563C (T).     

X-ray-induced chromosome aberrations in mouse dictyate oocytes. I. Time and dose relationships.

Structural chromosome aberrations were analyzed in superovulated metaphase-I oocytes of the mouse, Mus musculus, at various times after a single acute dose of 200 R of X-rays. The aberrations seen were of the chromatid type, i.e., chromatid interchanges, isochromatid deletions and chromatid deletions. The aberration frequency was low during the interval 24 h to 5 days between irradiation and ovulation; peak frequency was reached when irradiation was given 14 days prior to ovulation. A dose-response study was made 14 days prior to ovulation at doses of 50, 100, 200, 300 and 400 R. A curve of these data indicated that a significant two-track component was present for both interchanges and deletions. Centromere staining revealed that symmetrical and asymmetrical interchanges occurred at approximately equal frequency and also that the asymmetrical equivalent of crossing-over was induced at a measurable frequency.     

Cell survival and radiation induced chromosome aberrations

Summary Existing mathematical formulations to predict the frequency of radiation induced chromosome aberrations in 2nd post-irradiation division are based on the Poisson distribution [3, 4]. Meanwhile several studies have shown that intercellular distributions exist, deviating from Poisson. In the present study a modified model was developed which permits the application of empirical distributions. Transmission and survival parameters of aberrations can be iteratively computed. A general formula was derived for the calculation of cell survival from 1st to 2nd division.     

Monitoring natural vegetation for herbicide-induced chromosomal aberrations

The average frequency of spontaneous mitotic chromosome aberrations, determined in 12 weed species growing under natural conditions, was 0.4%. Herbicides induced significant increases in this frequency in five species, Ambrosia artemisiifolia L., Pastinaca sativa L., Solidago canadensis L., Solidago nemoralis Ait., and Vicia cracca L. The auxin herbicides — 2,4-D, picloram, and 2,4-D + 2,4,5-T — induced a larger proportion of lagging chromosomes and a smaller proportion of fragmented chromosomes than found among spontaneous aberrations. The non-selective herbicides, simazine and diuron, produced multipolar spindles that were not observed among untreated cells. Clastogenic effects of nonselective herbicides in Pastinaca sativa were short-lived, giving highest frequencies of aberrant cells in June and July and lower aberration rates in August and September. The opposite trend was found in untreated flower buds of this species, suggesting that the spontaneous aberration rate is higher in flower buds from older plants.     

The stage of chromosome duplication in the cell cycle as revealed by X-ray breakage and 3H-thymidine labeling

By means of combined experiments of X-irradiation and ³H-thymidine labeling of the chromosomes which are in the phase of synthesis, and the subsequent analysis at metaphase on the autoradiographs of the chromosomal damage induced during interphase, it was shown that in somatic cells from a quasi-diploid Chinese hamster line cultured in vitro the chromosomes change their response to radiation from single (chromosome type aberrations) to double (chromatid type aberrations) in late G₁. These results are interpreted to indicate that the chromosome splits into two chromatids in G₁, before DNA replication. — By extending the observations at the second metaphase after irradiation, it was also seen that cells irradiated while in G₂ or late S when they reach the second post-irradiation mitosis still exhibit, beside chromosome type aberrations, many chromatid exchanges, some of which are labeled. Two hypotheses are suggested to account for this unexpected reappearance of chromatid aberrations at the second post-irradiation division. The first hypothesis is that they arise from half-chromatid aberrations. The second hypothesis, which derives from a new interpretation of the mechanisms of production of chromosome aberrations recently forwarded by Evans, is that they arise from gaps or achromatic lesions which undergo, as the cells go through the next cycle, a two-step repair process culminating in the production of aberrations.This work was supported in part by grant No. RH-00304 from the Division of Radiological Health, Bureau of State Services, Public Health Service, U.S.A.     

On time dependency of frequency and distribution of chromatid aberrations induced by mutagens with delayed effects. An interpretation

This paper provides a theoretical analysis of pecularities of both the frequency and intrachromosomal distribution of chromatid aberrations observed in the first post-treatment mitosis and induced by clastogenic agents showing delayed effects (S-phase dependent clastogens), as functions of recovery time. The theoretical deductions are based on the following facts: (1) DNA is the target of clastogen action. Lesions induced by clastogens showing delayed effects (e.g. mono- and polyfunctional alkylating agents, ultraviolet light) give rise to aberrations only after interference with the process(es) associated with DNA replication. (2) DNA replication occurs asynchronously with respect to the local involvement in replication of different chromatin regions and according to a highly ordered pattern. (3) Lesions may be removed from DNA (or otherwise modified) by repair processes prior to replication. The removal of lesions from DNA is a time-dependent function.Several possibilities are analysed (i.e. random or non-random distribution of DNA lesions, uniform or locally differing capacities of pre-replicative repair of lesions, uniform or locally differing rates of DNA synthesis) and the frequencies and distribution patterns of chromosome structural changes, as expressed in form of aberration yield-time curves, have been discussed. The theory presented in this paper offers a simple interpretation both of variations of aberration frequency and aberration distribution in dependence on the cell's position within the cell cycle during induction of lesions.It is shown that the intrachromosomal aberration distribution is non-random even if random distribution of lesions and uniform repair rates between chromosome regions replicating at different time periods during S are assumed. Non-random aberration distributions are a necessary consequence of at least two factors: (a) the temporal replication pattern, and (b) the repair activities acting prior to replication. Random distribution of aberrations is only to be expected for the most simplified situation (random distribution of lesions along the DNA and equal transformation probabilities of a given kind of lesion into aberrations) when no loss of lesions prior to replication takes place (no pre-replicative repair) and cells treated with the mutagen during G1 are analysed.     

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.