Interpreting chromosome aberration spectra.

Ionizing radiation can damage cells by breaking both strands of DNA in multiple locations, essentially cutting chromosomes into pieces. The cell has enzymatic mechanisms to repair such breaks; however, these mechanisms are imperfect and, in an exchange process, may produce a large-scale rearrangement of the genome, called a chromosome aberration. Chromosome aberrations are important in killing cells, during carcinogenesis, in characterizing repair/misrepair pathways, in retrospective radiation biodosimetry, and in a number of other ways. DNA staining techniques such as mFISH (multicolor fluorescent in situ hybridization) provide a means for analyzing aberration spectra by examining observed final patterns. Unfortunately, an mFISH observed final pattern often does not uniquely determine the underlying exchange process. Further, resolution limitations in the painting protocol sometimes lead to apparently incomplete final patterns. We here describe an algorithm for systematically finding exchange processes consistent with any observed final pattern. This algorithm uses aberration multigraphs, a mathematical formalism that links the various aspects of aberration formation. By applying a measure to the space of consistent multigraphs, we will show how to generate model-specific distributions of aberration processes from mFISH experimental data. The approach is implemented by software freely available over the internet. As a sample application, we apply these algorithms to an aberration data set, obtaining a distribution of exchange cycle sizes, which serves to measure aberration complexity. Estimating complexity, in turn, helps indicate how damaging the aberrations are and may facilitate identification of radiation type in retrospective biodosimetry.     

No increase in radiation-induced chromosome aberration complexity detected by m-FISH after culture in the presence of 5'-bromodeoxyuridine

The thymidine analogue, 5'-bromodeoxyuridine (BrdU), is a known mutagen that is routinely introduced into culture media for subsequent Harlequin stain analysis and determination of cell cycle status. Previously, we examined the induction of chromosome aberrations in human peripheral blood lymphocytes (PBL) known to be in their 1st cell division following exposure to a low dose (0.5 Gy, average one alpha-particle per cell) of high-LET alpha-particles. We found complex chromosome aberrations to be characteristic of exposure to high-LET radiation and suggested the features of complex exchange to reflect qualitatively the spatial deposition of this densely ionising radiation. To exclude the possibility that BrdU addition post-irradiation influenced the complexity of chromosomal damage observed by m-FISH, the effect of increasing BrdU concentration on aberration complexity was investigated. Comparisons between BrdU concentration (0, 10 and 40 microM) and between sham- and alpha-particle-irradiated PBL, were made both independently and in combination to enable discrimination between BrdU and high-LET radiation effects. Aberration type, size, complexity and completeness were assessed by m-FISH, and the relative progression through cell division was evaluated. We found no evidence of any qualitative difference in the complexity of damage as visualised by m-FISH but did observe an increase in the frequency of complex exchanges with increasing BrdU concentration indicative of altered cell cycle kinetics. The parameters measured here are consistent with findings from previous in vitro and in vivo work, indicating that each complex aberration visualised by m-FISH is characteristic of the structure of the high-LET alpha-particle track and the geometry of cell irradiated.     

Radiation-induced chromosome aberrations: insights gained from biophysical modeling

Enzymatic misrepair of ionizing-radiation-induced DNA damage can produce large-scale rearrangements of the genome, such as translocations and dicentrics. These and other chromosome exchange aberrations can cause major phenotypic alterations, including cell death, mutation and neoplasia. Exchange formation requires that two (or more) genomic loci come together spatially. Consequently, the surprisingly rich aberration spectra uncovered by recently developed techniques, when combined with biophysically based computer modeling, help characterize large-scale chromatin architecture in the interphase nucleus. Most results are consistent with a picture whereby chromosomes are mainly confined to territories, chromatin motion is limited, and interchromosomal interactions involve mainly territory surfaces. Aberration spectra and modeling also help characterize DNA repair/misrepair mechanisms. Quantitative results for mammalian cells are best described by a breakage-and-reunion model, suggesting that the dominant recombinational mechanism during the G(0)/G(1) phase of the cell cycle is non-homologous end-joining of radiogenic DNA double strand breaks. In turn, better mechanistic and quantitative understanding of aberration formation gives new insights into health-related applications.     

The effect of functional inactivation of TP53 by HPV-E6 transformation on the induction of chromosome aberrations by gamma rays in human tumor cells

The influence of expression of TP53 (formerly known as p53) on the induction of chromosome aberrations by gamma rays was examined in an isogenic pair of human tumor cell lines where TP53 expression was normal or inactivated by human papillomavirus (HPV) type 16 E6 expression. Plateau-phase cultures were exposed to 0-8 Gy gamma rays and then either immediately released by subculture or held for 24 h prior to subculture and subsequent cytogenetic analysis. Aberration frequency was determined only in cells entering their first mitosis after irradiation, and cells were sampled over a 48-h period to include cells whose progression into mitosis was delayed. While aberration frequencies were similar at early harvest times, there was evidence for a subpopulation of more heavily damaged cells in the E6-transformed cells that cycled into late mitosis. Holding cells noncycling for 24 h to allow repair of potentially lethal damage eliminated this subpopulation of more heavily damaged cells. The E6-transformed cells also had higher levels of chromatid-type aberrations and sister chromatid exchanges, consistent with an additional defect in kinetics of repair of base damage that is associated with the E6 transformation. Holding cells noncycling for 24 h eliminated the elevated levels of chromatid-type aberrations and sister chromatid exchanges. These studies demonstrate that E6 transformation of human tumor cells will influence both the frequency and types of chromosome aberrations observed after radiation exposure, and that these effects are related to the expression of potentially lethal damage.     

The action of N-methyl-N-nitrosourea on non-established human cell lines in vitro. II. Non-random distribution of chromatid aberrations in diploid and Down's cells.

Chromatid gaps, breaks and aberrations involved in interchanges induced by N-methyl-N-nitrosourea (MNU) were found non-randomly distributed on individual chromosomes and chromosome segments (G bands) both in human diploid fibroblasts with trisomy 21 cultured in vitro. Aberration events were located exclusively in pale G bands. Considering cells in the first post-treatment mitosis, the pattern of aberration distribution, as revealed by the position of hot spots, varied with recovery time and was different in diploid and Down's cells. In comparison with diploid cells, the X chromosomes of Down's cells were not involved in aberrations. Despite the higher aberration frequencies of Down's cells, the number of hot spots and the proportion of aberrations located in hot spots were not increased in this cell type. Therefore, the increased chromosomal sensitivity to MNU of Down's cells does not reflect an increased sensitivity of special chromosomes or chromosome sites.     

Comparing DNA damage-processing pathways by computer analysis of chromosome painting data.

Chromosome aberrations are large-scale illegitimate rearrangements of the genome. They are indicative of DNA damage and informative about damage processing pathways. Despite extensive investigations over many years, the mechanisms underlying aberration formation remain controversial. New experimental assays such as multiplex fluorescent in situ hybridyzation (mFISH) allow combinatorial "painting" of chromosomes and are promising for elucidating aberration formation mechanisms. Recently observed mFISH aberration patterns are so complex that computer and graph-theoretical methods are needed for their full analysis. An important part of the analysis is decomposing a chromosome rearrangement process into "cycles." A cycle of order n, characterized formally by the cyclic graph with 2n vertices, indicates that n chromatin breaks take part in a single irreducible reaction. We here describe algorithms for computing cycle structures from experimentally observed or computer-simulated mFISH aberration patterns. We show that analyzing cycles quantitatively can distinguish between different aberration formation mechanisms. In particular, we show that homology-based mechanisms do not generate the large number of complex aberrations, involving higher-order cycles, observed in irradiated human lymphocytes.     

In vivo cytogenetic studies on mice exposed to ethylene dibromide

The pesticide, ethylene dibromide (EDB), was evaluated with in vivo cytogenetic assays to determine its genotoxicity. CD1 male mice were exposed to EDB through intraperitoneal injections. Bone marrow cells isolated from femora were analyzed for sister-chromatid exchange (SCE), chromosome aberration and micronucleus formation. The results showed that only certain concentrations of EDB tested caused a slight but significant increase in SCEs and chromosome aberrations. However, these increases were not dose-related. No increase in the polychromatic erythrocytes with micronuclei was observed following EDB exposure. Also, EDB did not cause cell-cycle delay in comparison with controls. Thus, it appears that EDB is not an effective genotoxic agent in vivo in mice.     

The Production of Chromosome Aberrations in Various Mammalian Cells by Triethylenemelamine

The cytogenetic effects of triethylenemelamine (TEM) were studied using five different mammalian tissues. Treatments of 0.1 and 0.2 mg/kg TEM on differentiating mouse spermatogonia and bone marrow cells showed no significant differences in the frequency of chromosomal aberrations produced in these two tissues. At higher doses, however, the sensitivites of the two tissues appear to be different. The frequency of aberrations varies with time after treatment, with the greatest amount occurring at the latter fixation times. Results of an experiment on primary spermatocytes indicated a correlation between the frequency of chromosome aberrations and DNA replication. Human peripheral leukocytes were utilized in an attempt to clarify the cell-stage specificity of TEM-induced chromosome aberrations. Cultures were treated with TEM prior to PHA stimulation (G0), as well as various time intervals after stimulation (late G,1 S, and G2). The most sensitive stages of the cell cycle to aberration induction were later G1 and S, with chromatid aberrations the predominant type. A very low yield of chromosome damage was observed with the G0 and G1 treated stages. The experiments described tend to support the view that TEM is most effective at inducing aberrations when an intervening round of DNA replication has occurred.     

Studies on radiation-induced chromosome aberrations in mouse spermatocytes. I. Stage specificity and dose-response relationships of chromosome aberrations induced in mouse primary spermatocytes following X-irradiation

The cytological analysis of chromosome aberrations induced at diplotene, mid-pachytene, zygotene and leptotene stages following X-irradiation was performed at diakinesis-metaphase I in mouse spermatocytes. The dose-response relationships fitted well to linear equations for deletion-type aberrations at each stage, and to linear-quadratic equations for exchange-type aberrations at all stages except for leptotene. The radiosensitivity to chromosome aberration induction tended to increase gradually with progression through synaptic and post-synaptic stages, diplotene being the most sensitive. Chromatid exchanges were hardly observed at leptotene, the aberrations being mainly isochromatid fragments. On the contrary, chromatid exchanges and isochromatid deletions were mainly observed at later stages (zygotene-diplotene). The specificity of chromosome aberration induction in primary spermatocytes might be influenced by chromatin organization and chromosomal configuration peculiar to meiotic cells.     

Aberrant and multiaberrant (rogue) cells in peripheral lymphocytes of Hodgkin's lymphoma patients after chemotherapy

We analyzed spontaneous chromosome lesions in peripheral lymphocytes cultured from Hodgkin's lymphoma (HL) patients before and after cytostatic chemotherapy. The mean aberration frequency was significantly higher in HL patients after chemotherapy (7.20+/-0.58 per 100 metaphases) than in non-treated HL patients (4.80+/-0.54), and in non-treated patients than in healthy subjects (2.12+/-0.13). In lymphocytes of HL patients, who received chemotherapy, we found, in addition to ordinary aberrant cells, a large number of multiaberrant (or rogue) cells, i.e. metaphases carrying multiple (at least four) chromosome-type exchange aberrations. Rogue cells were found in 15 out of 18 chemotherapeutically treated HL patients (in total, 60 rogue cells per 5,568 scored cells), whereas in 30 non-treated patients only 1 rogue cell was found (per 4,988 scored cells). No correlation was found between the yield of rogue cells and the aberration frequency in ordinary aberrant cells. Aberration spectra (ratios of chromatid- to chromosome-type aberrations and of breaks to exchanges) were essentially different in ordinary aberrant and multiaberrant cells. These data, as well as analysis of cellular distributions of aberrations, implied independent induction of chromosome damage in ordinary aberrant and rogue cells. Analysis of aberration patterns in diploid and polyploid rogue metaphases belonging to the first, second, and third in vitro division indicated that rogue cells could be formed both in vivo and in vitro, and could survive at least two rounds of in vitro replication, given blocked chromosome segregation. These results suggested that formation of rogue cells, unlike ordinary aberrant cells, was triggered by events other than direct DNA and/or chromosome lesions. A hypothesis regarding disrupted apoptosis as a candidate mechanism for rogue cell formation seems to be most suitable for interpretation of our data. Cultured lymphocytes of chemotherapeutically treated HL patients may represent a model system for further examination of the multiaberrancy phenomenon.     

A cytogenetic investigation of DNA rereplication after hydroxyurea treatment: implications for gene amplification

Although the mechanisms leading to gene amplification are poorly understood, it has recently been proposed that the initial event of amplification is the rereplication of a variable, but relatively large, amount of the genome within a single cell cycle. We sought evidence for rereplication of DNA as a basis for gene amplification through two cytogenetic techniques: differential staining for sister-chromatid exchange analysis and premature chromosome condensation. Synchronized Chinese hamster ovary cells were incubated continuously with bromodeoxyuridine and treated with hydroxyurea (HU) when cells were approximately 2 h into the S phase. After 6 h exposure to HU, the drug was removed and at 3 h intervals thereafter metaphase cells were collected and the chromosomes were stained by the fluorescence-plus-Giemsa procedure. No staining patterns consistent with rereplication of DNA were observed. Since HU causes cytogenetic damage, the premature chromosome condensation technique was used to determine the kinetics of chromosome damage after removal of HU. Extensive G₂ chromosome damage within 1 h after removal of HU from the medium was found, although cesium chloride gradient analysis showed that there was no rereplication of DNA during this time. Contrary to a previous report, these results provide no evidence that incubation of cells with HU during S phase induces rereplication of DNA within a single cell cycle. The results observed are consistent with the hypothesis that drug-induced aberrations and the subsequent abnormal segregation of chromosomal fragments are the first steps in the process that leads to gene amplification in drug-treated mammalian cells.     

Analysis of restriction enzyme-induced DNA double-strand breaks in Chinese hamster ovary cells by pulsed-field gel electrophoresis: implications for chromosome damage.

Restriction enzymes can be electroporated into mammalian cells, and the induced DNA double-strand breaks can lead to aberrations in metaphase chromosomes. Chinese hamster ovary cells were electroporated with PstI, which generates 3' cohesive-end breaks, PvuII, which generates blunt-end breaks, or XbaI, which generates 5' cohesive-end breaks. Although all three restriction enzymes induced similar numbers of aberrant metaphase cells, PvuII was dramatically more effective at inducing both exchange-type and deletion-type chromosome aberrations. Our cytogenetic studies also indicated that enzymes are active within cells for only a short time. We used pulsed-field gel electrophoresis to investigate (i) how long it takes for enzymes to cleave DNA after electroporation into cells, (ii) how long enzymes are active in the cells, and (iii) how the DNA double-strand breaks induced are related to the aberrations observed in metaphase chromosomes. At the same concentrations used in the cytogenetic studies, all enzymes were active within 10 min of electroporation. PstI and PvuII showed a distinct peak in break formation at 20 min, whereas XbaI showed a gradual increase in break frequency over time. Another increase in the number of breaks observed with all three enzymes at 2 and 3 h after electroporation was probably due to nonspecific DNA degradation in a subpopulation of enzyme-damaged cells that lysed after enzyme exposure. Break frequency and chromosome aberration frequency were inversely related: The blunt-end cutter PvuII gave rise to the most aberrations but the fewest breaks, suggesting that it is the type of break rather than the break frequency that is important for chromosome aberration formation.     

Mechanisms of chromosomal aberration production II. Aberrations induced by 5-bromodeoxyuridine and visible light

We have allowed synchronized V79B Chinese hamster tissue culture cells to incorporate 5-bromodeoxyuridine (BUdR) during one DNA synthetic (S) period of the cell cycle and then determined chromosomal aberration yields induced by illumination of the cells with visible light during the succeeding pre- and post-DNA-synthetic (G₁and G₂) phases of the cell cycle. At the level used, BUdR by itself induces no aberrations. Illumination during the G₁ phase following incorporation induces aberrations of the chromatid type, but none of the chromosome type. All types of chromatid aberrations are induced, including isochromatid deletions and exchange types. In contrast, when cells are illuminated during the immediately following G₂ phase, large numbers of achromatic lesions and chromatic deletions are seen at the first post-illumination mitosis, but no isochromatid deletions and few exchange-type aberrations occur. When G₂-illuminated cells are examined in their second mitosis, however, chromatid aberrations of all types are again seen.

These results are interpreted within the “repair” model of chromosomal aberration production by UV light presented earlier³. The model assumes that the vertebrate chromosome is mononeme, consisting of but a single DNA double helix during the prereplication G₁ phase. The initial lesions induced by illumination of BUdR-containing DNA are believed to be single-chain breaks, and the observation that G₁ illumination produces only chromatid-type aberrations is taken as additional evidence for the mononeme chromosome. Conversion of single-chain breaks into double chain breaks through the action of a single-strand nuclease is postulated to account for the production of chromatid deletions at the first mitosis of G₂-illuminated cells. The action of this enzyme, plus a recombinational or post-replication repair mechanism, are postulated to account for the production of isochromatid deletions in G₁-illuminated cells. A rapid decline in achromatic lesion frequency with increasing time between G₂ illumination and fixation of the cells is considered evidence for rapid rejoining of most of the initial chain breaks.     

Revision of th distribution of chromosome aberrations induced by chemical mutagens using the BUDR label

Cell distribution was analysed with the help of the BrDU label for the number of chromosome aberrations and breaks induced by one-center (thiophosphamide and phosphamide) and two-center (dipine and fotrine) mutagens at the stage G0 in the Ist mitosis of human lymphocytes harvested at different times of culturing (from 56 to 96 h). The comparison was made between the type of aberration distribution in cells and the dependence of their frequency on the harvesting point for various mutagens. Poisson aberration distribution in cells for two-center mutagens was found to correspond to their constant frequency observed at different times of harvesting. On the other hand, for one-center mutagens, a geometrical distribution of chromosome breaks corresponded to an exponential decrease in their frequency in time. It is suggested that two-center chemical mutagens and ionizing radiation cause largely short-live damages which are realized into chromosome aberrations rather quickly (during one cell cycle). One-center mutagens, however, cause such damages that the probability of their transformation into chromosome aberrations is decreasing rather slowly in time, under the exponential law, and their realization into chromosome aberrations can occur in subsequent cell cycle.     

3-Aminobenzamide does not affect X-ray-induced cytogenetic damage in G0 human lymphocytes

The inhibition of poly(ADP-ribose) polymerase by 3-aminobenzamide (3AB) has been reported to have very different effects on X-ray-induced chromosome aberrations in G0 human lymphocytes. One group of investigators observed a 2-3-fold increase in the yield of rings, dicentrics and chromosome breaks after X-irradiation and 3AB treatment, whereas another group found that 3AB had no effect on X-ray-induced chromosome aberrations. To resolve this discrepancy, we repeated the experiments as described by both groups and found no effect of 3 mM or 5 mM 3AB on the frequency of chromosome aberrations induced by either 1 Gy or 2 Gy of X-rays. Furthermore, we found no effect of 3AB on X-ray-induced aberration yields in C-banded prematurely condensed chromosome preparations from unstimulated human lymphocytes. These results indicate that poly(ADP-ribose) polymerase is not involved in the repair of cytogenetic damage in G0 human lymphocytes.     

The cytogenetic effects of aflatoxin and gamma-rays on human leukocytes in vitro

Chromosomes from human leukocyte cultures in vitro were treated with γ-rays (200 R), aflatoxin (50 μg/ml, dissolved in dimethyl sulfoxide (DMSO)) and with a combination of both. At the time of treatment (48 h) cells were in all stages of interphase but G₁ cells were evidently predominant. All types of chromosome aberration were observed. Frequencies of chromosome-type aberrations were much higher than those of chromatid type after γ-ray treatment, but these types of chromosome aberration did not differ greatly when the cultures were treated with aflatoxin. Apparently the cytogenetic effect of aflatoxin was delayed longer than was that of irradiation. The present data also suggest the additive effect of γ-rays and aflatoxin in the combined treatment.     

Molecular cytogenetics in the study of cutaneous T-cell lymphomas (CTCL)

The etiology of primary cutaneous T-cell lymphomas (CTCL) has been poorly understood. CTCL patients show a large variety of non-clonal and clonal chromosome aberrations, but no specific aberration has been found until recently. This review describes cytogenetic and molecular cytogenetic findings and their relevance to diagnostics and etiology in two of the most common forms of CTCL, mycosis fungoides (MF) and the leukemic CTCL, Sézary syndrome (SS).     

Chromosomal Instability Parameters in the Population Affected by Nuclear Explosions at the Semipalatinsk Nuclear Test Site

A population genetic survey of 149 persons who were born and have permanently lived in the contaminated zones of the Semipalatinsk region has been performed. A cytogenetic study has demonstrated that the frequency of aberrant cells is 1.7–3 times higher than control parameters. The total frequencies of chromosome aberrations are 3.43 ± 0.48, 3.1 ± 0.3, 1.8 ± 0.2, and 1.15 ± 0.17 aberrations per 100 cells in the populations of the extreme radiation risk (ERR), maximum radiation risk (MaxRR), minimum radiation risk (MinRR), and control zones, respectively. The high chromosome aberration rate in all three zones of radiation risk has been detected mainly due to radiation-induced chromosome markers, including paired fragments (1.3 ± 0.2, 0.94 ± 0.13, and 0.43 ± 0.06 per 100 cells, respectively), dicentric and ring chromosomes (0.44 ± 0.04, 0.45 ± 0.07, and 0.11 ± 0.02 per 100 cells, respectively), and stable chromosome aberrations (0.74 ± 0.16, 0.8 ± 0.1, and 0.63 ± 0.13 per 100 cells, respectively). The qualitative spectra of the cytogenetic lesions observed in these groups indicate a mutagenic effect of ionizing radiation on chromosomes in the populations studied.     

Genotoxic effects of malathion: an organophosphorus insecticide, using three mammalian bioassays in vivo

The genotoxic effects of malathion was evaluated using chromosome aberration, sister chromatid exchange (SCE) and sperm abnormality assays in mice. All the three acute doses (2.5, 5 and 10mg/kg) of malathion tested in the present study, induced significant dose-dependent increase in the frequency of chromosome aberrations and sperm abnormalities, but did not affect the total sperm count. The highest acute dose induced a >12-fold increase in the frequency of chromosome aberrations, two-fold increase in the frequency of SCEs and four-fold increase in the frequency of sperms with abnormal head morphology following intraperitoneal (i.p.) exposure. Further, a significant increase in the frequency of SCEs was observed, but the increase was not dose-dependent. At higher doses, malathion induced a moderate delay in cell cycle as evident from the increase in average generation time (AGT). The present findings suggest that technical grade malathion is a potent genotoxic agent and may be regarded as a potential germ cell mutagen also.     

Rearrangements of chromosome region 12q13----q15 in pleomorphic adenomas of the human salivary gland (PSA)

Nine of 40 pleomorphic salivary gland adenomas (PSAs) showed clonal aberrations of chromosome 12, with a breakpoint at 12q13----q15. The cytogenetic findings in these cases and those of nine additional cases reported in the literature suggest that this type of aberration is a primary change directly involved in the genesis of PSA.