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.     

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.     

Microdosimetry and chromosome aberrations: Effects of 230 keV neutrons on Vicia faba chromosomes

Physical energy deposition events have been related to sub-nuclear cytological events (chromosomal changes) in metaphases sequentially accumulated from the latter part of the cell cycle of Vicia faba. 230 keV neutrons produce about 0.4 recoil protons per late interphase nucleus per rad with the majority of protons travelling 1 to 2 microns from their origin, depositing energy at around 90 keV per micron. The frequency of induced aberrations is basically linear with dose, though varying through consecutive cell sampling periods because of differential induced mitotic delay. Distributions of chromosomal aberrations and total cytological events are overdispersed in relation to the Poisson distribuyion indicating that some proton recoils produce multiple events. When gaps and aberrations within chromosomes and multiple aberrations between chromosomes, are considered as discrete events, distributions follow Poisson expectations. About 40% of proton recoils result in observable cytological change. The highly energetic proton recoils (～90 keV per micron) which can induce multiple events are the ones most likely to produce effects which result in cell death. The sphere of influence of the proton recoils is probably adequately estimated from their range (～1 to 2 μm) since it seems compatible with the spatial proximity of the initial components of the resultant chromosome aberrations.     

Frequency of chromosome aberrations induced in human peripheral lymphocytes by in vitro 60Co gamma quanta at doses of 1 to 5 Gr. in an analysis of the 1st-division cells at different fixation times

The quantitative analysis of chromosomal aberrations in the first division cells of 50-, 54-, 58-, 52- and 66-hour peripheral blood lymphocytes cultures of healthy donors was performed after irradiation in vitro with 60Co gamma-quantums at doses 1--5 Gy. Cells of the first division were identified by a differential staining of sister chromatid method using 5-bromdeoxyuridine. No significant differences in frequencies of aberrant cells and aberrations of chromosomal type were found between cultures fixed at different times. The distribution of dicentrics in cells did not differ from the Poisson distribution regardless of fixation times and doses. On the basis of these findings it is concluded that chromosomes of human peripheral blood lymphocytes passing the cell cycle at different rates have approximately equal radiosensitivity.     

Number of aberrations per cell as a parameter of chromosome instability. 2. Comparative analysis of the factors of different nature

The average number of aberrations per aberrant cell was concluded to carry out information on chromosome instability peculiarities induced by different mutagens as it was shown in our previous work. The purpose of the current study was to present comparative analysis of intercellular distribution of number of aberrations and their theoretical approximations. Distribution of numbers of aberrations per cell in Allium cepa L. and Allium fistulosum L. root tip cells induced by different mutagenic factors (gamma-irradiation, thiotepa, formaldehyde and seed aging) have been studied. The results were approximated to theoretical Poisson, geometric and negative binomial distributions. The intercellular distribution of aberrations did not correspond to any of the used theoretical distributions when A. cepa seeds were gamma-irradiated. There was some, but not regular, accordance with theoretical distributions when chemical mutagens thiotepa in A. cepa and formaldehyde in A. fistulosum and seed aging in both species were evaluated. During seed aging frequency of aberrant cells increased more quickly in A. fistulosum in comparison with A. cepa.     

Chromosomal aberrations and sister-chromatid exchanges in Chinese hamster cells treated in vitro with hexavalent chromium compounds.

Chinese hamster cells (CHO line) were treated in vitro for 30--39 h with hexavalent chromium compounds (K2Cr2O7 and Na2CrO7), at concentrations ranging from 0.1 to 1.0 microgram of Cr6+ per ml, in medium containing BUdr. Chromosomal aberrations and sister-chromatid exchanges were scored on BUdr-labelled 2nd division metaphases, collected at the end of treatment and stained with Giemsa. Treatment with mitomycin C 0.009--0.030 microgram/ml) was carried out as a control for the responsiveness of the cell system to chromosomal damage. Both chromium compounds induced marked mitotic delays. Chromosomal aberrations were increased about 10-fold by exposure to Cr6+ (1.0 microgram/ml). The principal aberrations observed were single chromatid gaps, breaks and interchanges, whose frequencies increased proportionally to the concentration of chromium. Dicentric chromosomes, isochromatid breaks, chromosome and chromatid rings were also induced. The frequenyc of sister-chromatid exchanges was hardly doubled 30 h after exposure to Cr6+ at 0.3 microgram/ml, whereas it was trebled 39 h after treatment, in the cells whose division cycle had been slowed down by chromium.     

Effect of offspring distribution on population survival

The survival probabilities of newly-formed colonies of organisms arising from the branching process formulation of individual reproduction are examined. Six types of 2-parameter discrete offspring distributions common to mathematical ecology are compared with respect to survival of newly formed colonies of offspring. It is found that the survival value of the distribution can be rank ordered in the following descending order: modified Poisson (highest) Neyman A, geometric Poisson, Pólya-Aeppli, negative binomial and the modified geometric. Causal factors for these differences and practical implications of these results are discussed.     

Calculation of the radiation-induced delay in cell division during a study of the induction of chromosome aberrations (the theoretical and experimental bases of the approach)

The possible influence of a radiation-induced delay of cell division on the yield of chromosome aberrations has been analyzed theoretically. The analysis is based on the assumption that the delay is caused by the damages that are realized in the mitosis as chromosome aberrations. It has been shown that the distributions of cells, that are registered by the number of chromosome aberrations, are a function of time of cell fixation and may substantially differ from actual disturbance. There is a correlation between the registered and actual disturbances which is determined by the kinetics of the first mitosis of nonirradiated cells and the degree of the radiation-induced division delay. Some qualitative sequels of the proposed model are in agreement with the experimental data.     

Analysis of intercellular distributions of chromatid aberrations.

59 intercellular distributions of chemically induced and spontaneous chromatid aberrations were analyzed for goodness of fit in respect of the Poisson (PD), the geometrical (GD), and the negative binomial distributions (NBD). The data are excellently described by the NBD. This distribution can be deduced from a model out of the queueing theory and is based on the hypothesis of restitution. Estimators are obtained for the ratio of restitution and induction processes involved in the origin of chromosomal aberrations.     

Age and chromosome nondisjunction]

The parental age in 77 families of Down syndrome (DS) children with the known origin of extra chromosome 21 and in 12 families of DS children resulting from de novo translocation (more probable than not in 2 meiotic division) was studied. It was shown that when nondisjunction occurred in the 1st meiotic division, both in oogenesis (n = 30) and spermatogenesis (n = 12), mean parental ages and age distributions were different from that of control (400 couples with normal children). The mean age and age distribution were found to differ from control when nondisjunction occurred in the 2nd meiotic division of oogenesis (n = 19). On the basis of our information and the previously published data, lack of the effect of parental age on chromosome segregation in the Ist meiosis may be inferred. It is chromatid disjunction in the 2nd meiosis which is more probably age-dependent. The reasons preventing elucidation of real associations are under debate.     

Poisson adjacency distributions in genome comparison: multichromosomal, circular, signed and unsigned cases

The number of common adjacencies of genetic markers, as a measure of the similarity of two genomes, has been widely used as indicator of evolutionary relatedness and as the basis for inferring phylogenetic relationships. Its probability distribution enables statistical tests in detecting whether significant evolutionary signal remains in the marker order. In this article, we derive the probability distributions of the number of adjacencies for a number of types of genome--signed or unsigned, circular or linear, single-chromosome or multichromosomal. Generating functions are found for singlechromosome cases, from which exact counts can be calculated. Probability approaches are adopted for multichromosomal cases, where we.nd the exact values for expectations and variances. In both cases, the limiting distributions are derived in term of numbers of adjacencies. For all unsigned cases, the limiting distribution is Poisson with parameter 2; for all signed cases, the limiting distribution is Poisson with parameter (1/2).     

Computational model of chromosome aberration yield induced by high- and low-LET radiation exposures

We present a computational model for calculating the yield of radiation-induced chromosomal aberrations in human cells based on a stochastic Monte Carlo approach and calibrated using the relative frequencies and distributions of chromosomal aberrations reported in the literature. A previously developed DNA-fragmentation model for high- and low-LET radiation called the NASARadiationTrackImage model was enhanced to simulate a stochastic process of the formation of chromosomal aberrations from DNA fragments. The current version of the model gives predictions of the yields and sizes of translocations, dicentrics, rings, and more complex-type aberrations formed in the G(0)/G(1) cell cycle phase during the first cell division after irradiation. As the model can predict smaller-sized deletions and rings (<3 Mbp) that are below the resolution limits of current cytogenetic analysis techniques, we present predictions of hypothesized small deletions that may be produced as a byproduct of properly repaired DNA double-strand breaks (DSB) by nonhomologous end-joining. Additionally, the model was used to scale chromosomal exchanges in two or three chromosomes that were obtained from whole-chromosome FISH painting analysis techniques to whole-genome equivalent values.     

The meiotic behaviour of a haploid pearl millet

Meiosis was studied in one haploid plant of pearl millet, obtained from twin seedlings. Apparent pairing resulted in up to three bivalent associations at pachytene. At diakinesis and metaphase I associations of two, three or four chromosomes were observed. The frequency distribution of bivalents at metaphase followed a truncated Poisson distribution, suggesting that the bivalents were random pairs. They were considered to be pseudo-bivalents. Univalents varied in number from three to seven and they formed s-s and e-g associations. The s-s and e-s associations were random associations since their frequency distributions also followed a truncated Poisson distribution. A bipolar spindle was observed in a large number of PMC's but in a few cases two unipolar spindles were observed. The anaphase I distribution of the chromosomes deviated from abinomial distribution. Laggards were observed at telophase I. The dyads varied in size and in number of chromosomes. After the second division cell wall formation often failed to take place in one or in both the dyads, resulting in the formation of 2 to 4 microspores and microspores with two nuclei. The pollen grains varied in size and number of chromosomes. The plant was completely sterile.     

Chromosome aberrations in workers of nuclear-power plants

The frequencies of chromosome aberrations in 135 workers from nuclear-power plants were compared with those in 135 age-matched controls. A total of 135,000 cells was scored. The frequencies of dicentric chromosome were 1.67 × 10⁻³ in the exposed group and 0.49 × 10⁻³ in the control group and those of chromosome-type deletion were 3.33 × 10⁻³ and 1.10 × 10⁻³, respectively. The frequencies of all types of chromosome aberrations in the exposed subjects were higher than those in the control group, but no significant trend of dose-dependent increase was observed when only the exposed group were considered. Poisson regression analysis, with both exposed and control included, showed that there was a significant association of chromosome aberration with radiation dose and the duration of work, but not with age, smoking habit and alcohol intake. It was also found that recent exposure to radiation, within the last 5 years, had contributed more to the observed chromosome aberration than earlier exposure.     

A Comparison of Generalized Poisson Distribution for Modeling Chromosome Aberrations

Various models have been developed for modeling the distribution of chromosome aberrations in the literature (e. g. Consul, 1989). Generalized Poisson distribution is among the popular ones. The parameters of this distribution provide meaningful interpretation of the induction mechanism of the chromosome aberrations. In this article, we apply several estimation methods to estimate the generalized Poisson parameters for fitting the number of chromosome aberrations under different dosages of radiations. The methods compared are moment, maximum likelihood, minimum chi-square, weighted discrepancy and empirically weighted rates of change. Our study suggests that the empirically weighted rates of change method results in smallest Mean Square Error and Mean Absolute Error for most dosages of radiations. The data used for this comparison are from Janardan and Schaeffer (1977).     

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.     

PHOTOCONTROL OF THE ORIENTATION OF CELL DIVISION IN ADIANTUM. I. EFFECTS OF THE DARK AND RED PERIODS IN THE APICAL CELL OF GAMETOPHYTES

When protonemata of Adiantum capillus-veneris L. which had been grown filamentously under continuous red light were transferred to continuous white light, the apical cell divided transversely twice, but the 3rd division was longitudinal. An intervening period of darkness lasting from 0 to 90 hr either between the 1st and the 2nd cell division or between the 2nd and the 3rd one did not affect the number of protonemata in which the 3rd cell division was longitudinal. The insertion of red light instead of darkness greatly decreased the percentage of 1st longitudinal divisions occurring at the 3rd division, and increased the number of transverse divisions. Fifty percent reduction of induction of 1st longitudinal division was caused by ca. 50 hr exposure to red light between 1st and 2nd division and by ca. 20 hr between 2nd and 3rd division, and total loss was induced by an exposure of ca. 100 hr or longer to red light in the former and by ca. 40 hr longer in the latter. Thus, by using an appropriate intervening dark period or exposure to red light, the orientation and timing of cell division could be controlled in apical cell of the fern protonemata.     

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.     

Induction of chromosomal aberrations by camptothecin in root-tip cells of Vicia faba.

When root-tip cells of Vicia faba were exposed during early and middle interphase to camptothecin (Cpt), the aberrations obtained were exclusively of the chromatid type and tended to be localized in late replicating heterochromatic regions of the chromosomes. In these respects the clastogenic effect of Cpt resembles that of agents that act by an S-phase-dependent mechanism. In contrast to typical S-phase-dependent agents, Cpt produced lesions capable of giving rise to aberrations only in S-phase cells that were synthesizing DNA at the time of treatment. The dependence on ongoing DNA synthesis was suggested in autoradiographic experiments and by the fact that the clastogenic effect of Cpt was strongly suppressed by hydroxyurea, an inhibitor of DNA synthesis. After Cpt treatments, there were many more cells with 3-12 aberrations and far fewer cells with 0, 1 or 2 aberrations than expected on the basis of a Poisson distribution. Cpt further differed from typical S-phase-dependent agents by being capable of inducing lesions in the G2 phase that give rise to chromosomal aberrations in the first mitosis after treatment. This effect was obtained at Cpt concentrations around 10 microM, whereas only 0.03 microM Cpt was required to produce chromatid aberrations in the S phase. Results of G2-phase experiments with Cpt and 5-fluorodeoxyuridine, an inhibitor of DNA synthesis, suggest that DNA synthesis is required for the clastogenic effect of Cpt not only during the S phase, but also during the G2 phase, although the DNA syntheses involved are both quantitatively and qualitatively different.     

Biophysical analysis of the dose-dependent overdispersion and the restricted linear energy transfer dependence expressed in dicentric chromosome data from alpha-irradiated human lymphocytes

Experimental data for the induction of dicentric chromosomes in phytohemagglutinin (PHA)-stimulated human T lymphocytes by ²⁴¹Am alpha-particles obtained by Schmid et al. have been analyzed in the light of biophysical theory. As usual in experiments with alpha-particles, the relative variance of the intercellular distribution of the number of aberrations per cell exceeds unity, and the multiplicity of the aberrations per particle traversal through the cell is understood as the basic effect causing this overdispersion. However, the clearly expressed dose dependence of the relative variance differs from the dose-independent relative variance predicted by the multiplicity effect alone. Since such dose dependence is often observed in experiments with alpha-particles, protons, and high-energy neutrons, the interpretation of the overdispersion needs to be supplemented. In a new, more general statistical model, the distribution function of the number of aberrations is interpreted as resulting from the convolution of a Poisson distribution for the spontaneous aberrations with the overdispersed distributions for the aberrations caused by intratrack or intertrack lesion interaction, and the fluctuation of the cross-sectional area of the cellular chromatin must also be considered. Using a suitable mathematical formulation of the resulting dose-dependent overdispersion, the mean number λ ₁ of the aberrations produced by a single particle traversal through the cell nucleus and the mean number λ ₂ of the aberrations per pairwise approach between two alpha-particle tracks could be estimated. Coefficient α of the dose-proportional yield component, when compared between ²⁴¹Am alpha-particle irradiation and ¹³⁷Cs gamma-ray exposure, is found to increase approximately in proportion to dose-mean restricted linear energy transfer, which indicates an underlying pairwise molecular lesion interaction on the nanometer scale. Received: 17 December 1996 / Accepted in revised form: 20 April 1997