mBAND analysis of chromosomal aberrations in human epithelial cells exposed to low- and high-LET radiation

Energetic heavy ions pose a potential health risk to astronauts who have participated in extended space missions. High-LET radiation is much more effective than low-LET radiation in the induction of biological effects, including cell inactivation, genetic mutations, cataracts and cancer. Most of these biological end points are closely correlated with chromosomal damage, which can be used as a biomarker for radiation damage. Multicolor banding in situ hybridization (mBAND) has proven to be highly useful for the study of intrachromosomal aberrations, which have been suggested as a biomarker of exposure to high-LET radiation. To investigate biological signatures of radiation quality and the complexity of intrachromosomal aberrations, we exposed human epithelial cells in vitro to (137)Cs gamma rays or iron ions (600 MeV/nucleon) and collected chromosomes using a premature chromosome condensation technique. Aberrations in chromosome 3 were analyzed using mBAND probes. The results of our study confirmed the observation of a higher incidence of inversions for high-LET radiation. However, detailed analysis of the inversion type revealed that both iron ions and gamma rays induced a low incidence of simple inversions. Half of the inversions observed in the low-LET-irradiated samples were accompanied by other types of intrachromosome aberrations, but few inversions were accompanied by interchromosome aberrations. In contrast, iron ions induced a significant fraction of inversions that involved complex rearrangements of both inter- and intrachromosome exchanges.     

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.     

Effect of the pairing gene Ph1 and premeiotic colchicine treatment on intra- and interchromosome pairing of isochromosomes in common wheat.

The analysis of the pattern of isochromosome pairing allows one to distinguish factors affecting presynaptic alignment of homologous chromosomes from those affecting synapsis and crossing-over. Because the two homologous arms in an isochromosome are invariably associated by a common centromere, the suppression of pairing between these arms (intrachromosome pairing) would indicate that synaptic or postsynaptic events were impaired. In contrast, the suppression of pairing between an isochromosome and its homologous chromosome (interchromosome pairing), without affecting intrachromosome pairing, would suggest that homologous presynaptic alignment was impaired. We used such an isochromosome system to determine which of the processes associated with chromosome pairing was affected by the Ph1 gene of common wheat-the main gene that restricts pairing to homologues. Ph1 reduced the frequency of interchromosome pairing without affecting intrachromosome pairing. In contrast, intrachromosome pairing was strongly reduced in the absence of the synaptic gene Syn-B1. Premeiotic colchicine treatment, which drastically decreased pairing of conventional chromosomes, reduced interchromosome but not intrachromosome pairing. The results support the hypothesis that premeiotic alignment is a necessary stage for the regularity of meiotic pairing and that Ph1 relaxes this alignment. We suggest that Ph1 acts on premeiotic alignment of homologues and homeologues as a means of ensuring diploid-like meiotic behavior in polyploid wheat.     

Differential sensitivity to x-ray of chromosomes of blood T-lymphocytes and B-and T-cell lines.

Normal T-lymphocytes, B-cell line (CCRF-SB) and T-cell line (CCRF-HSB-2) cells, all diploid in their chromosome constitution, were exposed in vitro to various doses of X-ray and analyzed at their first mitotic division for structural chromosome abnormalities. The irradiation effects were determined also by a viability test of the cells, using trypan blue dye. The irradiated T-cell line (CCRF-HSB-2) showed a remarkably high frequency of chromosome aberrations, including chromosome and chromatid deletions, chromatid exchanges, dicentrics, rings and acentric fragments. On the other hand, the chromosome aberrations observed in the irradiated B-cell line and normal T-lymphocytes consisted mainly of dicentrics, rings, deletions and acentric fragments; the frequency of chromosome and chromatid deletions was low as compared to that of the T-cell line. The cell viability test showed a singificantly higher percent reduction of viable cells at every dose of X-ray in the irradiated T-cell line than in the B-cell line or the normal T-lymphocytes. It is possible that the increased radiosensitivity of the T-cell line is related to the original malignant nature of the cells, which originated from the lymphocytes of a patient with acute lymphoblastic leukemia.     

Differential sensitivity to X-ray of chromosomes of blood T-lymphocytes and B- and T-cell lines

Summary Normal T-lymphocytes, B-cell line (CCRF-SB) and T-cell line (CCRF-HSB-2) cells, all diploid in their chromosome constitution, were exposed in vitro to various doses of X-ray and analyzed at their first mitotic division for structural chromosome abnormalities. The irradiation effects were determined also by a viability test of the cells, using trypan blue dye. The irradiated T-cell line (CCRF-HSB-2) showed a remarkably high frequency of chromosome aberrations, including chromosome and chromatid deletions, chromatid exchanges, dicentrics, rings and acentric fragments. On the other hand, the chromosome aberrations observed in the irradiated B-cell line and normal T-lymphocytes consisted mainly of dicentrics, rings, deletions and acentric fragments; the frequency of chromosome and chromatid deletions was low as compared to that of the T-cell line. The cell viability test showed a significantly higher percent reduction of viable cells at every dose of X-ray in the irradiated T-cell line than in the B-cell line or the normal T-lymphocytes. It is possible that the increased radiosensitivity of the T-cell line is related to the original malignant nature of the cells, which originated from the lymphocytes of a patient with acute lymphoblastic leukemia. Supported in part by USPHS grants CA-14413 and CA-16935.     

Mentha piperita (Linn.) leaf extract provides protection against radiation induced chromosomal damage in bone marrow of mice

Oral administration of M. piperita (1 g/kg body weight/day) before exposure to gamma radiation was found to be effective in protecting against the chromosomal damage in bone marrow of Swiss albino mice. Animals exposed to 8 Gy gamma radiation showed chromosomal aberrations in the form of chromatid breaks, chromosome breaks, centric rings, dicentrics, exchanges and acentric fragments. There was a significant increase in the frequency of aberrant cells at 6 hr after irradiation. Maximum aberrant cells were observed at 12 hr post-irradiation autopsy time. Further, the frequency of aberrant cells showed decline at late post-irradiation autopsy time. However, in the animals pretreated with Mentha extract, there was a significant decrease in the frequency of aberrant cells as compared to the irradiated control. Also significant increase in percentage of chromatid breaks, chromosome breaks, centric rings, dicentrics, exchanges, acentric fragments, total aberrations and aberrations/damaged cell was observed at 12 hr post-irradiation autopsy time in control animals, whereas Mentha pretreated irradiated animals showed a significant decrease in percentage of such aberrations. A significant decrease in GSH content and increase in LPO level was observed in control animals, whereas Mentha pretreated irradiated animals exhibited a significant increase in GSH content and decrease in LPO level but the values remained below the normal. The radioprotective effect of Mentha was also demonstrated by determining the LD(50/30) values (DRF = 1.78). The results from the present study suggest that Mentha pretreatment provides protection against radiation induced chromosomal damage in bone marrow of Swiss albino mice.     

Possible mechanisms of the occurrence of chromosome restructurings. IV. Chromosome restructurings in spontaneous mutagenesis

An attempt was undertaken to modify the spontaneous mutation process by varying its conditions in somatic cells of different species and tissues. The rate of chromosome aberrations and their types were studied in anaphase and metaphase. Under normal conditions, chromosome breaks were only found to occur. Breakage of chromosomes occurs during interphase, and as a result, acentric fragments are located outside the equatorial plate during metaphase. This process of chromosome breakage leads to elimination of some genetic material, without concomitant exchanges, and therefore, it has been named "elimination" process. Spontaneous chromosome mutagenesis manifesting itself at cytogenetic level was concluded to be an elimination process directed to elimination of a portion of chromatin from chromosomes. When the conditions of spontaneous mutagenesis are altered, in particular, by cardiovascular diseases in man, by partial inhibition of DNA repair in mice and pea cells, by transformation of Chinese hamster cells, upon ageing of pea seeds-qualitative changes in the chromosomal aberrations are registered, connected with the appearance of chromosome exchanges and acentric fragments situated within the equatorial plate during metaphase. These two types of chromosome aberrations are proposed to be considered as new criteria of pathology. A system of processes was suggested to exist, preventing the appearance of aberrations during mitosis, and it is supposed to be one of the most significant homeostatic systems.     

Analysis of chromosome aberrations in lymphocytes of long-term heavy smokers

We assessed the incidence of structural chromosome aberrations in 500 diploid first-division metaphases from 48-h lymphocyte cultures from each of 6 non-smokers and from 6 persons who had smoked a minimum of 1 pack of cigarettes per day for at least 20 years. Cytogenetic analyses of coded slides revealed a single dicentric chromosome with its accompanying fragment and two symmetrical chromatid exchanges in 3000 metaphases from the non-smokers. In contrast, 9 dicentric chromosomes, 8 translocations or inversions, and 7 chromatid exchanges were observed in 3000 metaphases from lymphocyte cultures from the 6 heavy smokers. A total of 13 metaphases having chromosome-type inter- or intra-changes was noted including 9 with a single aberration, and 4 with 2 or more. Our findings provide additional evidence of the in vivo clastogenicity of cigarette smoke in long-term heavy smokers, and further demonstrate that the distribution of chromosome-type exchange aberrations is overdispersed relative to that expected based on Poisson assumptions.     

Mapping by transduction of mutator gene mutH in Escherichia coliz

Spontaneous levels of structural chromosomal aberrations in human peripheral lymphocytes were studied cytogenetically in 49 female and 56 male subjects. With a total of 16 267 metaphase spreads examined, 191 cells were found to contain chromosomal aberrations, giving a rate of 1.17%. The rates of individual aberration types were as follows: chromatid fragments, 0.39%; chromosome fragments, 0.71%; dicentrics, 0.06%; symmtrical and asymmetrical chromatid exchanges, 0.01%; and rings, 0.006%. There were significant differences in aberration yields between females aged 20–29 yr. and 60–70 yr., whereas males aged 20–50 yr. showed no difference. The two sexes differed significantly in chromatid fragments, chromosome fragments and aberrant cells.     

The distribution of chromosome damage, non-reciprocal translocations and clonal aberrations in lymphocytes from Chernobyl clean-up workers

In this paper we determined whether the frequencies of translocations and insertions are proportional to chromosome size in peripheral blood lymphocytes from Chernobyl nuclear accident clean-up workers and healthy unexposed control subjects. The frequency of aberrations among chromosomes 1, 2 and 4 in both groups was found to be significantly different from the distribution expected on the basis of chromosome size, although the difference was only marginally significant in controls. We also determined whether differences exist in aberration frequencies measured by two scoring systems: the classical method, where reciprocal exchanges are scored as one event, and PAINT, where each break junction is scored as a single event. The two scoring systems gave highly correlated results which yielded an interpretable arithmetic relationship between frequency measurements using the two systems. Approximately 34% of all translocations were observed to be non-reciprocal, and cells bearing clones of abnormal cells were observed in 6 of 198 subjects (3.0%). Our results demonstrate that clones of abnormal cells and the presence of non-reciprocal translocations contribute to the non-proportional distribution of radiation-induced and spontaneous cytogenetic damage.     

The localisation of radiation induced chromosome aberrations in relation to the distribution of heterochromatin in Secale cereale

The distribution of two break chromosome exchanges (dicentrics and centric rings) following gamma or neutron irradiation of resting seeds of rye, Secale cereale, has been investigated. The localisation of heterochromatin in the terminal ends of the chromosomes of rye facilitates distinguishing aberrations involving heterochromatin from others. Dicentrics found in or near heterochromatic regions were about 5 times more frequent after gamma irradiation and about 2.5 times more after neutron irradiation, than expected on a random distribution. The implications of these findings in relation to aberration formation are discussed.     

DNA repair deficiency and BPDE-induced chromosomal alterations in CHO cells

The induction of chromosomal aberrations and sister chromatid exchanges by BPDE was evaluated in parental and different DNA repair deficient Chinese hamster ovary cell lines in order to elucidate the mechanisms involved in their induction. These included the parental line (AA8), nucleotide excision repair (UV4, UV5, UV61), base excision repair (EM9), homologous recombination repair (Irs1SF) and non-homologous end joining (V3-3) deficient ones. The ranking of different cell lines for BPDE-induced chromosome aberrations was: UV4, Irs1SF, UV5, UV 61, EM9, V3-3, and AA8 in a descending order. Cells deficient in NER and HRR were found to be very sensitive, indicating the importance of these pathways in the repair of lesions induced by BPDE. For induction of SCEs, HRR and BER deficient cells were refractory, whereas the other cell lines responded with a dose-dependent increase. The possible mechanisms involved in BPDE-induced chromosomal alterations are discussed.     

Chromosome chromatid rearrangements resulting from the mitotic crossing-over between sister-chromatids in ring chromosomes of Creptis capillaris

Summary All radiation-induced aberrations in dry seeds of Crepis capillaris are chromosome rearrangements. The main types of chromosome rearrangements in the above tests were asymmetrical and symmetrical exchanges, ring chromosomes and ring deletions. The majority of ring chromosomes is of a chromosomes type which brings about paired rings. Fig. 1 presents the mechanism of the production of the paired rings. In a number of cases the structure of rings proved to be quite unexpected. Among middle size rings single rings proved to occur in 18.8%, among microrings-1.9% cases. Somewhat fewer are presented by pairs of rings one inside the other. The large rings present complex figures made by tangled chromatids. Two rings make one due to mitotic crossing-over between sister-chromatids (Fig. 5). Double crossing-over would lead to the exchange of part of material between two independent rings or to one ring being thrust into the other due to different strand positions in two points of the exchange. Large rings is the provision of complicated exchanges.     

Chromosome painting for cytogenetic monitoring of occupationally exposed and non-exposed groups of human individuals

The suitability of a three-color fluorescence in situ suppression hybridization technique was examined for monitoring five different groups of individuals: 30 occupied in radiology, 26 occupied in nuclear medicine or radiation physics, 32 patients with breast cancer, 26 occupied with military waste disposal, all presumably exposed to low doses of radiation or chemical mutagens and a non-exposed control group (N=29). The average frequency of breaks constituting the various aberrations did not significantly differ between the groups of medical radiation appliers and the control group. However, breast tumor patients and military waste disposers, as groups, showed a higher aberration rate than did healthy controls. Stable rearrangements mainly characterized the groups of controls, tumor patients, and radiation appliers, while a higher proportion of unstable aberrations was found in the chemically exposed individuals. Individuals with an increased frequency of aberrations could be detected within each examined group, which clearly determined the average values of the whole group. With respect to interchromosomal distribution of the breakpoints constituting the found aberrations and the involvement of the labeled chromosomes in rearrangements, the observed values were very close to the expected ones in the controls. A rather similar trend of deviations from expectation was observed in all other groups. Chromosome 4 was slightly over-affected, while chromosome 2 was slightly underrepresented in all analyzed groups (except tumor patients). Rearrangements of the labeled chromosomes with the unlabeled ones exceeded expectation. In conclusion, chromosome painting if included in further attempts of human population monitoring will broaden the basis of argumentation with respect to health risks introduced by mutagen exposure.     

Analysis of the Chromosome Aberrations Induced by X-Rays in Somatic Cells of DROSOPHILA MELANOGASTER

A technique has been perfected for enabling good microscope preparations to be obtained from the larval ganglia of Drosophila melanogaster. This system was then tested with X-rays and an extensive series of data was obtained on the chromosome aberrations induced in the various stages of the cell cycle.-The analysis of the results obtained offers the following points of interest: (1) There exists a difference in radio-sensitivity between the two sexes. The females constantly display a greater frequency of both chromosome and chromatid aberrations. They also display a greater frequency of spontaneous aberrations. (2) In both sexes the overall chromosome damage is greater in cells irradiated in stages G(2) and G(1). These two peaks of greater radiosensitivity are produced by a high frequency of terminal deletions and chromatid exchanges and by a high frequency of dicentrics, respectively. (3) The aberrations are not distributed at random among the various chromosomes. On the average, the Y chromosome is found to be more resistant and the breaks are preferentially localized in the pericentromeric heterochromatin of the X chromosome and of the autosomes. (4) Somatic pairing influences the frequency and type of the chromosome aberrations induced. In this system, such an arrangement of the chromosomes results in a high frequency of exchanges and dicentrics between homologous chromosomes and a low frequency of scorable translocations. Moreover, somatic pairing, probably by preventing the formation of looped regions in the interphase chromosomes, results in the almost total absence of intrachanges at both chromosome and chromatid level.     

Effects of vanillin on sister-chromatid exchanges and chromosome aberrations induced by mitomycin C in cultured Chinese hamster ovary cells

Effects of vanillin on the induction of sister-chromatid exchanges (SCEs) and structural chromosome aberrations by mitomycin C (MMC) were investigated in cultured Chinese hamster ovary cells. Vanillin induced neither SCEs nor chromosome aberrations by itself. However, an obvious increase in the frequency of SCEs was observed when MMC-treated cells were cultured in the presence of vanillin. The effect of vanillin was S-phase-dependent. On the contrary, the frequency of cells with chromosome aberrations was significantly decreased by the post-treatment with vanillin at G2 phase.     

Chromosome-damaging activity of a ruthenium radiosensitizer, RuCl2 (DMSO)2 (4-nitroimidazole)2, in Chinese hamster ovary cells in vitro

The metal complex, RuCl2 (DMSO)2 (4-nitroimidazole)2, 1, which has hypoxic radiosensitizing properties, was examined for genotoxic activity, as measured by the in vitro induction of chromosome aberrations (chromatid breaks and chromatid exchanges) in Chinese hamster ovary (CHO) cells. A dose-dependent increase in the frequencies of metaphases with chromatid aberrations was observed for 1. Addition of S9 liver microsomal mixture and 1 to the cultured CHO cells did not alter the clastogenic activity noted for the complex itself. The clastogenic (chromosome damaging) activity of a precursor complex, cis-RuCl2(DMSO)4 and the ligand, 4-nitroimidazole (4-NO2-Im) were found to be less than that of 1 at corresponding concentrations. A comparison with two drugs used clinically with radiation, cis-dichlorodiammineplatinum(II) (cis-DDP) and misonidazole (miso), indicated that the clastogenic activity of 1 was similar to miso and much less than that of cis-DDP.     

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.     

Association between G2-phase block and repair of radiation-induced chromosome fragments in human lymphocytes.

We have studied the induction of chromosomal aberrations in human lymphocytes exposed in G0 to X rays or carbon ions. Aberrations were analyzed in G0, G1, G2 or M phase. Analysis during the interphase was performed by chemically induced premature chromosome condensation, which allows scoring of aberrations in G1, G2 and M phase; fusion-induced premature chromosome condensation was used to analyze the damage in G0 cells after incubation for repair; M-phase cells were obtained by conventional Colcemid block. Aberrations were scored by Giemsa staining or fluorescence in situ hybridization (chromosomes 2 and 4). Similar yields of fragments were observed in G1 and G2 phase, but lower yields were scored in metaphase. The frequency of chromosomal exchanges was similar in G0 (after repair), G2 and M phase for cells exposed to X rays, while a lower frequency of exchanges was observed in M phase when lymphocytes were irradiated with high-LET carbon ions. The results suggest that radiation-induced G2-phase block is associated with unrejoined chromosome fragments induced by radiation exposure during G0.     

Quantitative analyses of the induction of chromosome aberrations and sister-chromatid exchanges in human lymphocytes exposed to gamma-rays and mitomycin-C in combination

Most chemicals are S-dependent and are potent inducers of SCE, but do not produce chromosome-type aberrations in the first metaphases after exposure. Ionizing radiation, which is an S-independent agent, produces chromosome-type aberrations, especially dicentrics and rings, but inefficiently produces chromatid-type aberrations. A series of experiments has been performed to investigate whether cytogenetic damage induced by ionizing radiation (gamma-rays) might be assessed separately from that induced by the alkylating chemical, mitomycin C (MMC), when human lymphocytes were exposed to these 2 agents in combination. Whole-blood cultures of human lymphocytes in G0 phase were exposed to gamma-rays and MMC in combination or separately. Cytogenetic analyses were done for both chromosome aberrations (CA), analyzed in cultures incubated for 56 h without BrdUrd, and sister-chromatid exchanges (SCEs) in cultures incubated for 72 h with BrdUrd. The frequency of chromosome-type aberrations (dicentrics and rings) increased with increasing doses of gamma-rays from 0.5 to 4.0 Gy. The dose-response relationships were the same with or without concomitant treatment with MMC (10(-6) M). Although the SCE frequency increased with increasing doses of MMC, the increase was nearly the same as when cells were treated with both MMC and gamma-rays (2 Gy). There was no interaction between MMC and gamma-rays concerning these 2 endpoints.