The RBE of 30 kV X-rays for the induction of dicentric chromosomes in human lymphocytes

Summary The frequency of dicentric chromosomes induced by the irradiation of human lymphocytes inG ₀ phase was determined with hard (150 kV) and soft (30 kV) X-rays. When a linear-quadratic dose-effect relation was used to fit the experimental data, a significant linear contribution was found for 30 kV X-rays. The RBE of 30 kV compared with 150 kV X-rays approaches the value 3 at a 30 kV X-ray dose of 20 rad and decreases with increasing dose. From the results it may be concluded that chromatids with primary breaks, undergoing second order reactions and thus forming dicentric chromosomes, are produced by traversals of low-energy electrons through chromatin material.     

Unstable Chromosome Aberrations Do Not Accumulate in Normal Human Fibroblast after Fractionated X-Irradiation

We determined the frequencies of dicentric chromosomes per cell in non-dividing confluent normal human fibroblasts (MRC-5) irradiated with a single 1 Gy dose or a fractionated 1 Gy dose (10X0.1 Gy, 5X0.2 Gy, and 2X0.5 Gy). The interval between fractions was between 1 min to 1440 min. After the completion of X-irradiation, the cells were incubated for 24 hours before re-plating at a low density. Then, demecolcine was administrated at 6 hours, and the first mitotic cells were collected for 42 hours. Our study demonstrated that frequencies of dicentric chromosomes in cells irradiated with a 1 Gy dose at different fractions were significantly reduced if the fraction interval was increased from 1 min to 5 min (p<0.05, χ²-test). Further increasing the fraction interval from 5 up to 1440 min did not significantly affect the frequency of dicentric chromosomes. Since misrejoining of two independent chromosome breaks introduced in close proximity gives rise to dicentric chromosome, our results indicated that such circumstances might be quite infrequent in cells exposed to fractionated X-irradiation with prolonged fraction intervals. Our findings should contribute to improve current estimation of cancer risk from chronic low-dose-rate exposure, or intermittent exposure of low-dose radiation by medical exposure.     

Cytological effects of kepone on Chinese hamster cells

Cytological and cytotoxic effects of kepone on Chinese hamster cells (M3-1) were investigated. Cells treated with 2 micrograms, 4 micrograms, and 6 micrograms/ml of kepone did not show any morphological abnormalities. However, cytological observations showed that chromosome breaks, chromatid breaks, dicentric chromosomes, and chromosome interchanges were produced by these treatments. Cytotoxic studies revealed dose response and time response reactions to kepone. Cell toxicity was greater at the 30 micrograms/ml concentration, producing 100 percent cell death within 24 hours.     

Chromosome abnormalities in tuberous sclerosis

Summary In fibroblasts cultured from biopsies of the skin lesions of six patients with tuberous sclerosis (TS) there was a variable but consistent degree of karyotypic variation. Premature centromere disjunction (PCD) of all or part of the chromosomes, micronuclei, an increased incidence of breaks, dicentric chromosomes and the presence of polyploid metaphases were found in all cultures. The PCD was of the type encountered in Roberts syndrome and its frequency varied from 8% to 30%. In metaphases with PCD of one and of two chromosomes, the chromosome involved were identified, and chromosome 3 was involved 21 times among 59 chromosomes with PCD. Chromosome 3 tends to be preferentially involved in dicentric formation. In lymphocyte cultures from the same patients there were no metaphases with PCD, but there was a slight increase of breaks and the presence of dicentric chromosomes, also involving chromosome 3. Polyploid metaphases were increased in some of the cases. Karyotypic variation can be considered a cellular phenotypic characteristic of TS in fibroblasts cultured from the skin lesions, and its type indicates disturbances in the mechanics of centromere division and of chromosome distribution at cell division.     

Chromosomal aberrations in peripheral lymphocytes of patients treated with radium-224 for ankylosing spondylitis

The aim of this study was to investigate the in vivo frequency of chromosomal aberrations (primarily dicentric chromosomes and chromatid breaks) potentially induced by ²²⁴Ra -radiation in peripheral lymphocytes. The study was designed to serve as a cytogenetic analysis along with the therapeutic procedure of ankylosing spondylitis patients who were undergoing a treatment with ²²⁴Ra-chloride. The total administered activity was 10 MBq, and the treatment followed a schedule of 10 i.v. injections per week, each with a dose of 1 MBq of ²²⁴Ra. The calculation of absorbed doses delivered to the blood used the models suggested by the ICRP and yielded a value of 4.7 mGy/MBq. The frequency of chromosomal aberrations observed during the course of therapy was related to the blood dose. The frequency of dicentric chromosomes induced in vivo was found to agree well with the corresponding value of dicentrics induced in vitro. However—given that peripheral lymphocytes are in the cell cycles G₀ stage—an unexpected increase with dose in the yield of chromatid breaks was observed, with about 95% of them occurring in cells without any other chromosome-type aberrations. Reasons for the production of chromatid breaks are discussed.     

Time dependence of Elkind-type recovery in class B oocytes of Drosophila melanogaster.

Recovery from X-ray-induced damage in class B oocytes of Drosophila melanogaster was studied by the dose-fractionation technique. A total dose of 500 R was delivered either as a single exposure or as two fractions of 2000 R and 3000 R separated by increasing time intervals. The use of attached-X females made it possible to study simultaneously the induction of dominant lethals and of chromosome aberrations (detachments of the attached-X chromosome). The same repair kinetics were observed for sublethal damage and for the lesions leading to detachments. The time-response curves are of similar shape: a plateau is reached within 20 to 30 min and half of the repairable damage disappears in 5 to 7 min. It is concluded that the same type of X-ray-induced primary lesion in chromosomes is responsible for the induction of detachments and for dominant lethals. As primary lesions actual chromosome breaks or lesions leading to breaks and chromosome rearrangements are assumed.     

Interaction distance of primary lesions in the formation of dicentric chromosomes after irradiation of human lymphocytes with 3-MeV electrons in vitro

The rejoining distance for the formation of dicentric chromosomes in human lymphocytes is derived on the basis of microdosimetric concepts. For the formation of a dicentric chromosome, primary lesions produced by absorption events can interact within the nucleus over a distance of between 0.5 and 1.0 μ. The intercellular distribution of dicentric chromosomes is consistent with a Poisson distribution.     

Dicentric chromosomes and the inactivation of the centromere

Summary The origin and behavior of human dicentric chromosomes are reviewed. Most dicentrics between two non-homologous or two homologous chromosomes (isodicentrics), which are permanent members of a chromosome complement, probably originate from segregation of an adjacent quadriradial; such configurations are the result of a chromatid translocation between two nonhomologous chromosomes, or they represent an adjacent counterpart of a mitotic chiasma. The segregation of such a quadriradial may also give rise to a cell line monosomic for the chromosome concerned (e.g., a 45,X line). Contrary to the generally held opinion, isodicentrics rarely result from an isolocal break in two chromatids followed by rejoining of sister chromatids. In this case the daughter centromeres go to opposite poles in the next anaphase, and the resulting bridge breaks at a random point. This mechanism, therefore, leads to the formation of an isodicentric chromosome only if the two centromeres are close together, or if one centromere is immediately inactivated. Observations on the origin of dicentrics in Bloom syndrome support these conclusions. One centromere is permanently inactivated in most dicentric chromosomes, and even when the dicentric breaks into two chromosomes, the centromere is not reactivated. The appearance and behavior of the acentric X chromosomes show that their centromeres are similarly inactivated and not prematurely divided. Two Bloom syndrome lymphocytes, one with an extra chromosome 2 and the other with an extra chromosome 7, each having an inactivated centromere, show that this can also happen in monocentric autosomes.     

Radiation dosimetry by automatic image analysis of dicentric chromosomes.

A system for scoring dicentric chromosomes by image analysis comprised fully automatic location of mitotic cells, automatic retrieval, focus and digitization at high resolution, automatic rejection of nuclei and debris and detection and segmentation of chromosome clusters, automatic centromere location, and subsequent rapid interactive visual review of potential dicentric chromosomes to confirm positives and reject false positives. A calibration set of about 15,000 cells was used to establish the quadratic dose response for 60Co gamma-irradiation. The dose-response function parameters were established by a maximum likelihood technique, and confidence limits in the dose response and in the corresponding inverse curve, of estimated dose for observed dicentric frequency, were established by Monte Carlo techniques. The system was validated in a blind trial by analysing a test set comprising a total of about 8000 cells irradiated to 1 of 10 dose levels, and estimating the doses from the observed dicentric frequency. There was a close correspondence between the estimated and true doses. The overall sensitivity of the system in terms of the proportion of the total population of dicentrics present in the cells analysed that were detected by the system was measured to be about 40%. This implies that about 2.5 times more cells must be analysed by machine than by visual analysis. Taking this factor into account, the measured review time and false positive rates imply that analysis by the system of sufficient cells to provide the equivalent of a visual analysis of 500 cells would require about 1 h for operator review.     

Chk2 and P53 Regulate the Transmission of Healed Chromosomes in the Drosophila Male Germline

When a dicentric chromosome breaks in mitosis, the broken ends cannot be repaired by normal mechanisms that join two broken ends since each end is in a separate daughter cell. However, in the male germline of Drosophila melanogaster, a broken end may be healed by de novo telomere addition. We find that Chk2 (encoded by lok) and P53, major mediators of the DNA damage response, have strong and opposite influences on the transmission of broken-and-healed chromosomes: lok mutants exhibit a large increase in the recovery of healed chromosomes relative to wildtype control males, but p53 mutants show a strong reduction. This contrasts with the soma, where mutations in lok and p53 have the nearly identical effect of allowing survival and proliferation of cells with irreparable DNA damage. Examination of testes revealed a transient depletion of germline cells after dicentric chromosome induction in the wildtype controls, and further showed that P53 is required for the germline to recover. Although lok mutant males transmit healed chromosomes at a high rate, broken chromosome ends can also persist through spermatogonial divisions without healing in lok mutants, giving rise to frequent dicentric bridges in Meiosis II. Cytological and genetic analyses show that spermatid nuclei derived from such meiotic divisions are eliminated during spermiogenesis, resulting in strong meiotic drive. We conclude that the primary responsibility for maintaining genome integrity in the male germline lies with Chk2, and that P53 is required to reconstitute the germline when cells are eliminated owing to unrepaired DNA damage.     

Chromosomal anomalies induced by the organic phosphate pesticide guthion in Chinese hamster cells

Chromosomal anomalies associated with the use of the organic phosphate pesticide guthion were investigated in Chinese hamster cells (line CHO-K1). Most commonly observed were chromatid breaks and exchanges. Infrequently, mild failure of condensation, despiralization, secondary constriction, gaps, pulverization, ring and dicentric chromosomes were noted. The mean number of chromosome breaks per cell was significantly higher in treated cells than in control cells. Autoradiographic studies revealed that while higher dosages of the chemical (80–120 μg/ml) arrested cells and prevented their movement out of S phase, a lower dosage (60 μg/ml) caused a progression delay. In relation to the relative length of no. 1 and no. 2 chromosomes, no apparent difference existed in the incidence of total breaks between them. However, significant differences in the nonrandom distribution of breaks in no. 1 and no. 2 chromosomes indicated linear differentiation of breakage susceptibility along the chromosomes. An increased concentration of breaks was observed in the long arms of both no. 1 and no. 2 chromosomes. The experimental results suggest that guthion, as well as inducing chromosomal anomalies, may produce a viable mutant.     

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

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

Time course study of the chromosome-type breakage-fusion-bridge cycle in maize.

The B chromosome of maize has been used in a study of dicentric chromosomes. TB-9Sb is a translocation between the B and chromosome 9. The B-9 of TB-9Sb carries 60% of the short arm of 9. For construction of dicentrics, a modified B-9 chromosome was used, B-9-Dp9. It consists of the B-9 chromosome plus a duplicated 9S region attached to the distal end. In meiosis, fold-back pairing and crossing over in the duplicated region gives a chromatid-type dicentric B-9 that subsequently initiates a chromatid-type breakage-fusion-bridge cycle. In the male, it forms a single bridge in anaphase II of meiosis and at the first pollen mitosis. However, the cycle is interrupted by nondisjunction of the B centromere at the second pollen mitosis, which sends the B-9 dicentric to one pole and converts it from a chromatid dicentric to a chromosome dicentric. As expected, the new dicentric undergoes the chromosome-type breakage-fusion-bridge cycle and produces double bridges. A large number of plants with chromosome dicentrics were produced in this way. The presence of double bridges in the root cells of plants with a chromosome dicentric was studied during the first 10 wk of development. It was found that the number of plants and cells showing double bridges declined steadily over the 10-wk period. Several lines of evidence indicate that there was no specific developmental time for dicentric loss. "Healing" of broken chromosomes produced by dicentric breakage accounted for much of the dicentric loss. Healing produced a wide range of derived B-9 chromosomes, some large and some small. A group of minichromosomes found in these experiments probably represents the small end of the scale for B-9 derivatives.     

Spontaneous and radiation-induced chromosomal instability and persistence of chromosome aberrations after radiotherapy in lymphocytes from prostate cancer patients

The aim of the study was to compare the spontaneous and ex vivo radiation-induced chromosomal damage in lymphocytes of untreated prostate cancer patients and age-matched healthy donors, and to evaluate the chromosomal damage, induced by radiotherapy, and its persistence. Blood samples from 102 prostate cancer patients were obtained before radiotherapy to investigate the excess acentric fragments and dicentric chromosomes. In addition, in a subgroup of ten patients, simple exchanges in chromosomes 2 and 4 were evaluated by fluorescent in situ hybridization (FISH), before the onset of therapy, in the middle and at the end of therapy, and 1 year later. Data were compared to blood samples from ten age-matched healthy donors. We found that spontaneous yields of acentric chromosome fragments and simple exchanges were significantly increased in lymphocytes of patients before onset of therapy, indicating chromosomal instability in these patients. Ex vivo radiation-induced aberrations were not significantly increased, indicating proficient repair of radiation-induced DNA double-strand breaks in lymphocytes of these patients. As expected, the yields of dicentric and acentric chromosomes, and the partial yields of simple exchanges, were increased after the onset of therapy. Surprisingly, yields after 1 year were comparable to those directly after radiotherapy, indicating persistence of chromosomal instability over this time. Our results indicate that prostate cancer patients are characterized by increased spontaneous chromosomal instability. This instability seems to result from defects other than a deficient repair of radiation-induced DNA double-strand breaks. Radiotherapy-induced chromosomal damage persists 1 year after treatment.     

Behavior of dicentric chromosomes in budding yeast

DNA double-strand breaks arise in vivo when a dicentric chromosome (two centromeres on one chromosome) goes through mitosis with the two centromeres attached to opposite spindle pole bodies. Repair of the DSBs generates phenotypic diversity due to the range of monocentric derivative chromosomes that arise. To explore whether DSBs may be differentially repaired as a function of their spatial position in the chromosome, we have examined the structure of monocentric derivative chromosomes from cells containing a suite of dicentric chromosomes in which the distance between the two centromeres ranges from 6.5 kb to 57.7 kb. Two major classes of repair products, homology-based (homologous recombination (HR) and single-strand annealing (SSA)) and end-joining (non-homologous (NHEJ) and micro-homology mediated (MMEJ)) were identified. The distribution of repair products varies as a function of distance between the two centromeres. Genetic dependencies on double strand break repair (Rad52), DNA ligase (Lif1), and S phase checkpoint (Mrc1) are indicative of distinct repair pathway choices for DNA breaks in the pericentromeric chromatin versus the arms.     

Telomere loss in somatic cells of Drosophila causes cell cycle arrest and apoptosis

Checkpoint mechanisms that respond to DNA damage in the mitotic cell cycle are necessary to maintain the fidelity of chromosome transmission. These mechanisms must be able to distinguish the normal telomeres of linear chromosomes from double-strand break damage. However, on several occasions, Drosophila chromosomes that lack their normal telomeric DNA have been recovered, raising the issue of whether Drosophila is able to distinguish telomeric termini from nontelomeric breaks. We used site-specific recombination on a dispensable chromosome to induce the formation of a dicentric chromosome and an acentric, telomere-bearing, chromosome fragment in somatic cells of Drosophila melanogaster. The acentric fragment is lost when cells divide and the dicentric breaks, transmitting a chromosome that has lost a telomere to each daughter cell. In the eye imaginal disc, cells with a newly broken chromosome initially experience mitotic arrest and then undergo apoptosis when cells are induced to divide as the eye differentiates. Therefore, Drosophila cells can detect and respond to a single broken chromosome. It follows that transmissible chromosomes lacking normal telomeric DNA nonetheless must possess functional telomeres. We conclude that Drosophila telomeres can be established and maintained by a mechanism that does not rely on the terminal DNA sequence.     

Calculation of the dose-rate dependence of the decentric yield after Co gamma-irradiation of human lymphocytes.

The dose relation for dicentrics after in vitro Co gamma-irradiation of human lymphocytes with dose rates of 50 and 1.7 rad/min fit the linear-quadratic function y=alpha D + beta D2. Compared with 50 rad/min, after 1.7 rad/min alpha D was unchanged, whereas beta D2 was decreased by 36 percent. By applying the mean interaction time t approximate to 110 min for primary breaks, determined in an earlier experiment, and Lea's G-function, a theoretical dose relation for 1.7 rad/min can be calculated from the corresponding experimental curve. Thus, from calibration curves with conventional dose rates dose-effect curves for low dose rates can be derived without the need for time-consuming chromosome analyses.     

Role of Ku80-dependent end-joining in delayed genomic instability in mammalian cells surviving ionizing radiation

Ionizing radiation induces delayed destabilization of the genome in the progenies of surviving cells. This phenomenon, which is called radiation-induced genomic instability, is manifested by delayed induction of radiation effects, such as cell death, chromosome aberration, and mutation in the progeny of cells surviving radiation exposure. Previously, there was a report showing that delayed cell death was absent in Ku80-deficient Chinese hamster ovary (CHO) cells, however, the mechanism of their defect has not been determined. We found that delayed induction of DNA double strand breaks and chromosomal breaks were intact in Ku80-deficient cells surviving X-irradiation, whereas there was no sign for the production of chromosome bridges between divided daughter cells. Moreover, delayed induction of dicentric chromosomes was significantly compromised in those cells compared to the wild-type CHO cells. Reintroduction of the human Ku86 gene complimented the defective DNA repair and recovered delayed induction of dicentric chromosomes and delayed cell death, indicating that defective Ku80-dependent dicentric induction was the cause of the absence of delayed cell death. Since DNA-PKcs-defective cells showed delayed phenotypes, Ku80-dependent illegitimate rejoining is involved in delayed impairment of the integrity of the genome in radiation-survived cells.     

Chromosomal aberrations involving telomeres in BRCA1 deficient human and mouse cell lines

Cells defective in BRCA1 show genomic instability as evidenced by increased radiosensitivity, the presence of chromosomal abnormalities and the loss of heterozygosity at many loci. Reported chromosomal abnormalities in BRCA1 deficient cells include dicentric chromosomes. Dicentric chromosomes, in some cases, may arise as a result of end-to-end chromosome fusions, which represent signatures of telomere dysfunction. In this study we examined BRCA1 deficient human and mouse cells for the presence of chromosomal aberrations indicative of telomere dysfunction. We identified a lymphoblastoid cell line, GM14090, established from a BRCA1 carrier that showed elevated levels of dicentric chromosomes. Molecular cytogenetic analysis revealed that these dicentric chromosomes result from end-to-end chromosome fusions. The frequency of end-to-end chromosome fusions did not change after exposure of GM14090 cells to bleomycin but we observed elevated levels of chromosomal abnormalities involving interactions between DNA double strand breaks and uncapped telomeres in this cell line. We observed similar chromosomal abnormalities involving telomeres in the breast cancer cell line, HCC1937, homozygous for BRCA1 mutation. Finally, we analyzed mouse embryonic stem cells lacking functional Brca1 and observed the presence of telomere dysfunction following exposure of these cells to bleomycin. Our results reveal cytogenetic evidence of telomere dysfunction in BRCA1 deficient cells.     

Chromosome aberration in human lymphocytes after X-irradiation in vitro. II. Analysis of primary processes in the formation of dicentric chromosomes

The rejoining distance for the formation of dicentric chromosomes in human lymphocytes has been derived on the basis of microdosimetric concepts. For the formation of a dicentric chromosome, primary lesions produced by absorption events can interact within the nucleus over a distance of at least 1 μm. The dispersion of dicentrics is near to 1 and corresponds to a site number between 17 and 120.