In vivo cytogenetics: mammalian germ cells

This chapter summarizes the most relevant methodologies available for evaluation of cytogenetic damage induced in vivo in mammalian germ cells. Protocols are provided for the following endpoints: numerical and structural chromosome aberrations in secondary oocytes or first-cleavage zygotes, reciprocal translocations in primary spermatocytes, chromosome counting in secondary spermatocytes, numerical and structural chromosome aberrations, and sister chromatid exchanges (SCE) in spermatogonia, micronuclei in early spermatids, aneuploidy in mature sperm. The significance of each methodology is discussed. The contribution of novel molecular cytogenetic approaches to the detection of chromosome damage in rodent germ cells is also considered.     

Metformin (dimethyl-biguanide) induced DNA damage in mammalian cells

Metformin (dimethyl-biguanide) is an insulin-sensitizing agent that lowers fasting plasma-insulin concentration, wherefore it's wide use for patients with a variety of insulin-resistant and prediabetic states, including impaired glucose tolerance. During pregnancy it is a further resource for reducing first-trimester pregnancy loss in women with the polycystic ovary syndrome. We tested metformin genotoxicity in cells of Chinese hamster ovary, CHO-K1 (chromosome aberrations; comet assays) and in mice (micronucleus assays). Concentrations of 114.4 μg/mL and 572 μg/mL were used in in vitro tests, and 95.4 mg/kg, 190.8 mg/kg and 333.9 mg/kg in assaying. Although the in vitro tests revealed no chromosome aberrations in metaphase cells, DNA damage was detected by comet assaying after 24 h of incubation at both concentrations. The frequency of DNA damage was higher at concentrations of 114.4 μg/mL. Furthermore, although mortality was not observed in in vitro tests, the highest dose of metformin suppressed bone marrow cells. However, no statistically significant differences were noted in micronuclei frequencies between treatments. In vitro results indicate that chronic metformin exposure may be potentially genotoxic. Thus, pregnant woman undergoing treatment with metformin should be properly evaluated beforehand, as regards vulnerability to DNA damage.     

Chemically induced aneuploidy in mammalian cells in culture

Our objectives were to assess whether there exist useful aneuploidy tests in vitro, to identify chemicals that showed potential for mitotic aneuploidy induction, and to recommend some features of suitable protocols for such testing. From over 100 papers we selected 24 for review. The acceptable studies examined hyperdiploidy at metaphase, had concurrent negative controls with low background rates of hyperdiploidy, used a fixation time sufficient for cells to complete more than one cell cycle after treatment and had multiple dose levels with at least 100 cells scored per point. We judged that 12 compounds were positive, 7 inconclusive, and 4 negative with the reservation that 2 of the 4 compounds had not been tested up to toxic doses. Many of the positive compounds are also known to cause structural chromosome aberrations. We separately reviewed qualitative reports of 'C-mitotic' effects, anaphase lagging, multipolar mitoses, or altered DNA content, since these effects may sometimes by associated with aneuploidy induction. No well-validated in vitro aneuploidy assay exists, and much research is required to develop tests, perhaps using chromosome counts, DNA content, or effects on cell organelles necessary for mitosis. In test protocol development we should carefully consider choice of cell sample size, use of in vitro metabolic activation systems, and selection of doses, especially with regard to the problem of whether cytotoxic concentrations should be used.     

Chromosomal abnormalities and the morphology of mouse sperm heads.

In the mouse, numerous mutagens, teratogens and carcinogens have been shown to induce marked elevations in the fraction of sperm with head shape abnormalities. Since carcinogens and teratogens may act by causing genetic damage, a likely explanation of these results is that the sperm abnormalities are also caused by genetic damage. There are two more or less distinct classes of genetic damage, chromosomal aberrations and point mutations. In this paper, we provide evidence, that in general, chromosomal aberrations are not responsible for causing abnormally shaped sperm. Chromosomal aberrations could have caused abnormal sperm morphology in a number of ways. One possibility was that the mere presence of a translocated chromosome within the germ cell led to the malformation of the sperm head. A second possibility was that chromosomal imbalance, i.e., aneuploidy, duplications or deficiencies, within the spermatid or haploid cells caused abnormalities in shape. We tested these hypotheses by measuring the level of abnormally shaped sperm in mice homozygous and heterozygous for 24 various reciprocal and Robertsonian translocations. The diploid cells of these mice are known to be chromosomally balanced, containing translocated chromosomes. A predictable proportion of their gametes are, however, chromosomally unbalanced and carry translocated chromosomes. It was found that the levels of sperm abnormalities in these mice were convincingly unrelated to the levels predicted by any of the above hypotheses. Based on these results it seems that sperm abnormalities in mice are not due to the mere presence of translocated chromosomes in germ cells and also not due to chromosomal aneuploidy or duplication-deficiencies of chromosomal segments in the spermatid during development of the sperm.     

Spontaneous chromosomal aberrations in Fanconi anaemia,ataxia telangiectasia fibroblast and Bloom's syndrome lymphoblastoid cell lines as detected by conventional cytogenetic analysis and fluorescence in situ hybridisation (FISH) technique

Several primary and transformed human cell lines derived from cancer prone patients are employed routinely for biochemical and DNA repair studies. Since transformation leads to some chromosomal instability a cytogenetic analysis of spontaneous chromosome aberrations in fibroblast cell lines derived from patients with Fanconi anaemia (FA), ataxia telangiectasia (AT), and in lymphoblastoid cell lines derived from patients with Bloom's syndrome (BS), was undertaken. Unstable aberrations were analysed in Giemsa stained preparations and the chromosome painting technique was used for evaluating the frequencies of stable aberrations (translocations). In addition, the frequency of sister-chromatid exchanges (SCEs) was determined in differentially stained metaphases. The SV40-transformed fibroblasts from these cell lines have higher frequencies of unstable aberrations than the primary fibroblasts. In the four lymphoblastoid cell lines derived from BS patients higher frequencies of spontaneously occurring chromosomal aberrations in comparison to normal TK6wt cells were also evident. The frequency of spontaneously occurring chromosome translocations was determined with fluorescence in situ hybridisation (FISH) and using DNA libraries specific for chromosomes 1, 2, 3, 4, 7, 8, 11, 14, 19, 20 and X. The translocation levels were found to be elevated for primary FA fibroblasts and lymphoblastoid cells derived from BS patients in comparison with control cell lines, hetero- and homozygote BS cell lines not differing in this respect. The SV40-transformed cell lines showed very high frequencies of translocations independent of their origin and almost every cell contained at least one translocation. In addition, clonal translocations were found in transformed control TK6wt and AT cell lines for chromosomes 20 and 14, respectively. The spontaneous frequencies of SCEs were similar in transformed fibroblasts derived from normal individuals and AT patients, whereas in SV40-transformed FA cells these were higher (4-fold). Among cell lines derived from BS patients, heterozygote lines behaved like control, whereas in homozygote cell lines very high frequencies of SCEs (about 12-fold) were evident.     

A detailed analysis of chromosomal changes in heritable and non-heritable retinoblastoma

Summary Full cytogenetic analysis of 27 different retinoblastoma tumors is presented. Gross aneuploidy of chromosome arms 6p and 1q were very common, being observed in 15/27 and 21/27 tumors, respectively. However, we found that chromosome 13 was rarely missing: only 3/27 had a detectable monosomy affecting 13q14. Monosomy of chromosome 13 by small deletion or rearrangement was also not observed in any of 12 retinoblastoma tumor lines analyzed detail at the 300–400 chromosome band level. A novel observation in retinoblastoma was the discovery of non-random translocations at three specific breakpoints, 14q32 (4/12), 17p12 (5/12), and 10q25 (3/12). Genomic rearrangements similar to those described involving C-myc in Burkitt lymphoma 14q+ cells could not be demonstrated in the four 14q+ retinoblastoma lines using molecular techniques, and a probe mapping to the site implicated to have an activating role in lymphoma. These data suggest that there is a target for rearrangement at 14q32 but it is not the same sequence used in some Burkitt lymphomas. Two other breakpoints (2p24 and 8q24) coincided with the mapped position of cellular oncogenes, but also failed to show a molecular rearrangement with the oncogene probes. The breakpoints, 10q25 and 17p12, are constitutional fragile sites which may predispose these regions to act as acceptors of translocations in malignant cells. One line had double minute chromosomes, and was the only one of 16 (6%) tested with the N-myc probe which had an amplification. Different tumors from single patients with multifocal heritable retinoblastoma showed independent karyotype evolution. Unilateral non-heritable tumors exhibited a high level of karyotype stability throughout both in vivo and in vitro growth. The various common patterns of aneuploidy and translocations probably confer an early selective advantage to malignant cells, rather than induce malignant transformation.     

Effect of linear energy transfer (LET) on the complexity of alpha-particle-induced chromosome aberrations in human CD34+ cells

The aim of this study was to assess the relative influence of the linear energy transfer (LET) of alpha particles on the complexity of chromosome aberrations in the absence of significant other differences in track structure. To do this, we irradiated human hemopoietic stem cells (CD34+) with alpha particles of various incident LETs (110-152 keV/microm, with mean LETs through the cell of 119-182 keV/microm) at an equi-fluence of approximately one particle/cell and assayed for chromosome aberrations by mFISH. Based on a single harvest time to collect early-division mitotic cells, complex aberrations were observed at comparable frequencies irrespective of incident LET; however, when expressed as a proportion of the total exchanges detected, their occurrence was seen to increase with increasing LET. Cycle analysis to predict theoretical DNA double-strand break rejoining cycles was also carried out on all complex chromosome aberrations detected. By doing this we found that the majority of complex aberrations are formed in single non-reducible cycles that involve just two or three different chromosomes and three or four different breaks. Each non-reducible cycle is suggested to represent "an area" of finite size within the nucleus where double-strand break repair occurs. We suggest that the local density of damage induced and the proximity of independent repair areas within the interphase nucleus determine the complexity of aberrations resolved in metaphase. Overall, the most likely outcome of a single nuclear traversal of a single alpha particle in CD34+ cells is a single chromosome aberration per damaged cell. As the incident LET of the alpha particle increases, the likelihood of this aberration being classed as complex is greater.     

X-ray- and mitomycin C (MMC)-induced chromosome aberrations in spermiogenic germ cells and the repair capacity of mouse eggs for the X-ray and MMC damage

Chromosome aberrations induced at the first-cleavage metaphase of eggs fertilized with sperm recovered from spermiogenic cells which had been X-irradiated and treated with mitomycin C (MMC) at various stages were observed using in vitro fertilization and embryo culture technique. Furthermore, the repair capacity of the fertilized eggs for X-ray- and MMC-induced DNA damage which was induced in the spermiogenic cells and retained in the sperm until fertilization was investigated by analysis of the potentiation effects of 2 repair inhibitors, 3-aminobenzamide (3AB) and caffeine on the yield of chromosome aberrations. The frequency of chromosome aberrations observed in the eggs fertilized with sperm recovered from the early spermatid to late spermatocyte stage with X-irradiation of 4 Gy (16-20 days after X-irradiation) was markedly higher than that in the eggs fertilized with sperm recovered from spermatozoa to late spermatid stage (0-8 days after X-irradiation). The induced chromosome aberrations predominantly consisted of chromosome-type aberrations, the main type being chromosome fragment followed by chromosome exchange through all the spermiogenic stages. On the other hand, a high frequency of chromosome aberrations was not induced through all the stages with MMC treatment of 5 mg/kg. The remarkable potentiation effects of 3AB and caffeine were found in the eggs fertilized with sperm recovered from almost all the spermiogenic stages after X-irradiation. In the MMC treatment, a remarkable caffeine effect was observed occasionally in mid-early spermatids to late spermatocytes where a large amount of MMC damage could be induced. These results suggest that the large amount of DNA lesions induced in spermiogenic cells by X-rays and MMC persist as reparable damage until sperm maturation and are effectively repaired in the cytoplasm of the fertilized eggs.     

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.     

ATM modulates the loading of recombination proteins onto a chromosomal translocation breakpoint hotspot

Chromosome translocations induced by DNA damaging agents, such as ionizing radiation and certain chemotherapies, alter genetic information resulting in malignant transformation. Abrogation or loss of the ataxia-telangiectasia mutated (ATM) protein, a DNA damage signaling regulator, increases the incidence of chromosome translocations. However, how ATM protects cells from chromosome translocations is still unclear. Chromosome translocations involving the MLL gene on 11q23 are the most frequent chromosome abnormalities in secondary leukemias associated with chemotherapy employing etoposide, a topoisomerase II poison. Here we show that ATM deficiency results in the excessive binding of the DNA recombination protein RAD51 at the translocation breakpoint hotspot of 11q23 chromosome translocation after etoposide exposure. Binding of Replication protein A (RPA) and the chromatin remodeler INO80, which facilitate RAD51 loading on damaged DNA, to the hotspot were also increased by ATM deficiency. Thus, in addition to activating DNA damage signaling, ATM may avert chromosome translocations by preventing excessive loading of recombinational repair proteins onto translocation breakpoint hotspots.     

Genetic and epigenetic effects of environmental arsenicals

Environmental arsenic compounds and their methylated metabolites do not form adducts with DNA, but do cause oxidative DNA damage. Chromosome aberrations are seen at toxic concentrations. Genetic effects that occur at non-toxic concentrations include aneuploidy, comutagenesis (resulting from indirect effects on DNA repair), and delayed mutagenesis (probably secondary to aneuploidy and/or epigenetic effects). Effects of trivalent arsenicals on poly(ADP ribose) polymerase and P53 activation may mediate effects on DNA repair and aneuploidy. A growing literature points to the epigenetic effects of arsenic compounds in cells and in vivo. A review of the current literature on DNA methylation, histone modifications and microRNA effects is presented.     

High rates of "unselected" aneuploidy and chromosome rearrangements in tel1 mec1 haploid yeast strains

The yeast TEL1 and MEC1 genes (homologous to the mammalian ATM and ATR genes, respectively) serve partially redundant roles in the detection of DNA damage and in the regulation of telomere length. Haploid yeast tel1 mec1 strains were subcultured nonselectively for approximately 200 cell divisions. The subcultured strains had very high rates of chromosome aberrations: duplications, deletions, and translocations. The breakpoints of the rearranged chromosomes were within retrotransposons (Ty or delta-repeats), and these chromosome aberrations nonrandomly involved chromosome III. In addition, we showed that strains with the hypomorphic mec1-21 allele often became disomic for chromosome VIII. This property of the mec1-21 strains is suppressed by a plasmid containing the DNA2 gene (located on chromosome VIII) that encodes an essential nuclease/helicase involved in DNA replication and DNA repair.     

A correlative study of the genetic damage induced by chemical mutagens in bone marrow and spermatogonia of mice. III. 1,3-Bis (2-chloroethyl)-3-nitrosourea (BCNU)

Cytogenetic damage induced by various concentrations of BCNU was evaluated by determining the frequencies of (a) micronuclei in polychromatic erythrocytes of bone marrow, (b) chromatid aberrations in bone marrow, (c) chromatid aberrations in spermatogonia, and (d) reciprocal translocations induced in spermatogonia and scored in spermatocytes. The order of sensitivity for the four parameters tested was: micronuclei greater than chromatid aberrations in bone marrow greater than aberrations in spermatogonia greater than translocations in spermatocytes, a situation similar to that found in an earlier study with CNU-ethanol. When the effect of concentration of the chemical was taken into consideration there was no correlation among the four parameters tested, so that information on induced frequencies of one parameter does not have predictive value for the frequencies of the others. A comparison of the results obtained with the bifunctional BCNU and the mono-functional CNU-ethanol at equal concentrations indicated that BCNU had a similar or a lesser clastogenic effect than did CNU-ethanol. In an experiment in vitro the situation was different in that BCNU was more effective than CNU-ethanol.     

Comparison of the rates of aneuploidy occurrence in quiescent and dividing cells of humans exposed to adverse environmental factors

Fluorescence in situ hybridization (FISH) was used to compare aneuploidy rates in four autosomes and two sex chromosomes in interphase nuclei of noncultivated (quiescent) and cultivated (induced to divide with phytohemagglutinin (PHA)) leukocytes in people engaged in nuclear-chemical industry and in a control group of people not exposed to mutagenic factors occupationally or at home. The overall rates of numerical chromosome aberrations for all of the six chromosomes studied showed little difference, although a higher rate of loss of the X- and Y-chromosomes was observed in the exposed group. In individuals exposed to several adverse environmental factors, the overall rate of numerical chromosome aberrations in cultivated cells after at least one DNA replication cycle exceeded that in noncultivated cells by 52% (P = 0.01), whereas only a trend for its increase was observed in the control group (23%, P = 0.25). Thus, the effect of adverse environmental factors in humans caused more than a twofold increase in the difference between the rates of aneuploid cells in cultivated and non-cultivated leukocytes in the exposed group as compared to control. It is conjectured that cell division is accompanied by the expression of potential damage of mitotic chromatid segregation apparatus accumulated in vivo. These defects, realized during cell division, bring about numerical chromosome aberrations.     

The characterization and transmissibility of chromosome aberrations induced in peripheral blood lymphocytes by in vitro alpha-particle radiation

Peripheral blood lymphocytes were irradiated in vitro with (213)Bi alpha particles at doses of 0, 10, 20, 50, 100, 200 and 500 mGy. Chromosome analysis was performed on 47-h cultures using single-color fluorescence in situ hybridization (FISH) to paint chromosomes 1, 3 and 5. The whole genome was analyzed for unstable aberrations to derive aberration frequencies and determine cell stability. The dose response for dicentrics was 33.60 +/- 0.47 x 10(-2) per Gy. A more detailed analysis revealed that the majority of aberrations scored as dicentrics were part of complex/multiple aberrations, with the proportion of cells containing complexes increasing with dose. Cells containing aberrations involving painted chromosomes (FISH aberrations) were further classified according to cell stability and complexity. The majority of cells with FISH aberrations were unstable. The proportion of aberrant FISH cells with complex/multiple aberrations ranged from 56% at 10 mGy to 89% at 500 mGy. A linear dose response for genomic frequencies of translocations in stable cells fitted the data from 0 to 200 mGy with a dose response of 7.90 +/- 0.98 x 10(-2) per Gy, thus indicating that they are likely to be observed in peripheral blood lymphocytes from individuals with past or chronic exposure to high-LET radiation. Comparisons with the dose response for low-LET radiation suggest an RBE of 13.6 for dicentrics in all cells and 3.2 for translocations in stable cells. Since stochastic effects of radiation are attributable to genetic changes in viable cells, translocations in stable cells may be a better measure when considering the comparative risks of different qualities of radiation.     

Molecular cytogenetic analysis of intergeneric chromosomal translocations between wheat (Triticum aestivum L.) and Dasypyrum villosum arising from tissue culture.

Fluorescence in situ hybridization (FISH) was applied with total genomic DNA extracted from Dasypyrum villosum (L.) Candargy as a probe to characterize chromosome translocations arising from tissue culture in hybrids of Triticum aestivum x (T. durum - D. villosum, amphiploid). Chromosome translocations between wheat and D. villosum occurred in callus cells at an average frequency of 1.9%. Translocations existed not only in callus cells but also in regenerants. Three plants with translocation chromosomes were characterized among 66 regenerants of T. aestivum 'Chinese Spring' x 'TH1W' and 'NPFP' x 'TH1'. One of them proved to be a reciprocal translocation with an exchange of about one third of a wheat chromosome arm with about one half of a chromosome arm of D. villosum. The breakpoints of the other two translocations were located at, or near centromeres. The results are similar for both callus cells and regenerants and provide further evidence that translocations take place in tissue culture. Other structural chromosomal changes, for example, fragments, telocentrics, dicentromeres, and deletions, as well as numerical alterations including aneuploidy and polyploidy were recorded both in callus cells and regenerants.     

A comparison of chromosomal radiosensitivities of somatic cells of mouse and man.

A cell culture technique for quantitative analysis of radiation-induced chromosome aberrations in somatic cells has been developed and used for the comparison of chromosomal sensitivity of skin cells of mouse and man to 60Co-gamma-rays. This includes culture of irradiated tissues or cells in culture in arginine and isoleucine-deficient medium and subsequent refeeding with complete medium (CM). With this technique, radiation-induced chromosome aberrations can be analyzed selectively in the cells exposed in G1 phase and recovered at their first post-irradiation mitosis. When tested on the human embryonic cells, the dicentric yield was essentially the same whether they were skin cells irradiated in silu or cultured cells at various in vitro passages irradiated in vitro. In contrast, when studied in the skin cells irradiated in silu, mouse embryos and newborns were insensitive to the induction of dicentrics. In young mice on day II however, the sensitivity was at a level comparable to that in human embryonic cells and it was intermediate on day 4. Such embryonic insensitivity of the mouse cells was rapidly lost during serial transfer in vitro; and, when tested at 4th or later subculture generations, mouse and human cells were equally sensitive to the induction of dicentrics. These results suggest that the chromosomal radiosensitivity is essentially the same for mouse and human cells but can be modified by some biological factors, possibly DNA repair mechanisms, which differ between species as well as among the states of differentiation of particular cell types. Special attention was paid to the parellelism between the age-dependent changes in the chromosomal, mutational and carcinogenic radiosensitivities in the mouse. If this parallelism can be carried over to man, human pre-natal irradiation will not present any reduced genetic hazards.     

Cytotoxic and genotoxic effect of inhibitor of vulcanisation N-cyclohexylthiophthalimide in a battery of in vitro assays

Mutagenicity of N-cyclohexylthiophthalimide (Duslin P) was tested first by the Ames test in the bacteria, Salmonella typhimurium. The negative results of the Ames test suggested that this compound does not induce mutations in the genome of S. typhimurium under the conditions used. To estimate the cytotoxicity of Duslin P to human cells, we measured cellular DNA and protein as well as cell proliferation, i.e., the mitotic index of treated and control cells. The genotoxic effects were assayed by two biochemical methods developed for detection of single-strand breaks of DNA in mammalian cells, i.e., by the alkaline single cell gel electrophoresis (comet assay) and by the DNA unwinding method, respectively. The DNA unwinding method showed that this compound did not induce DNA damage at concentrations < 7 micrograms/ml. Alkaline single cell gel electrophoresis revealed approximately double the level of DNA damage (in comparison to untreated control DNA) at a concentration of 2 micrograms/ml, which reduced proliferation to approximately 30%, and triple the level of DNA damage at higher concentrations (6 and 7 micrograms/ml), which inhibited completely both DNA synthesis and proteosynthesis. Cells with moderately damaged DNA were more common than cells with heavily damaged DNA. Parallel experiments with the strong mutagen and carcinogen MNNG showed that MNNG induced in cells a high level of DNA damage at concentrations which did not reduce the mitotic index or proteosynthesis, while DNA synthesis inhibited only partially. After treatment with MNNG, cells with heavily damaged DNA were more common than cells with moderately damaged DNA. Duslin P-treated VH10 cells were also tested cytogenetically, confirming that Duslin P induced neither chromosomal aberrations nor aneuploidy. We conclude that Duslin P has no mutagenic effect on bacteria, does not induce chromosomal aberrations and CREST positive or CREST negative micronuclei in human cells and induces only a small increase of DNA damage in human cells which is consistent with DNA fragmentation due to cell death.     

Anticlastogenic effect of Vitamin C on cisplatin induced chromosome aberrations in human lymphocyte cultures.

Vitamin C (ascorbic acid) is an antioxidant that can scavenge free radicals and protect cellular macromolecules, including DNA, from oxidative damage induced by different agents. The protective effect of Vitamin C on cisplatin induced chromosome aberrations has been determined in the human peripheral lymphocyte chromosome aberration test in vitro. The results of treatments with Vitamin C indicated that it statistically significantly decreases the number of chromosome aberrations and number of metaphases with aberrations induced with cisplatin, but it can not completely protect cells from damage. The test concentrations of Vitamin C (10 and 100 microg/ml) had a limited antimutagen effect on cisplatin (0.5 microg/ml), which can cause genetic damage through free radical mechanisms. The antimutagen effect included the anticlastogenic effect of Vitamin C and its ability to decrease the number of aneuploid mitoses. Vitamin C showed the most efficient anticlastogenic effect during simultaneous treatment with cisplatin. Also, Vitamin C reduced cell toxicity of cisplatin during simultaneous treatment.     

Effects of higher-order nuclear structure and Rad51 overexpression on radiation-induced chromosome rearrangements

The microchromosomes (MICs) in chicken DT40 lymphocytes are usually clustered in the center of the nucleus, whereas the macrochromosomes (MACs) are preferentially located toward the nuclear periphery. This compartmentalized architecture of the nucleus is associated with a low frequency of translocations between MICs and MACs after induction of DNA breaks by a radiation track(s). In contrast, the MICs in chick embryo fibroblasts (CEFs) tend to be located throughout the entire nuclear volume. The resulting side-to-side arrangement of MIC and MAC territories favors radiation-induced MIC/MAC translocations, which occur more frequently in CEF cells than MIC/MIC or MAC/MAC rearrangements. Collectively, our results suggest that preformed physical contacts are a prerequisite for the generation of chromosome rearrangements through recombinational repair of DNA damage. Cell type-specific higher-order nuclear organization may prevent or stimulate the formation of particular chromosome aberrations in pathology and evolution. Ectopic expression of the recombination protein Rad51 can protect cells from radiation-induced translocations. The repair activity of overexpressed Rad51 is more important for cells that are irradiated in S/G(2) phase than for cells in G(1) phase. Evidently, homologous recombination between sister chromatids of a replicated chromosome is more frequent than that between homologous or heterologous chromosomes during G(1) phase.