Frequency of mutagen-induced sister chromatid exchanges in the course of 3 cell cycles

The induction of SCE by fotrine (0.125 and 0.250 microgram/ml) and thiophosphamide (5 micrograms/ml) during the first three cell cycles was studied in the Chinese hamster cells. No increase in the SCE number was observed after treatment with thiophosphamide and fotrine at the G2 stage (the first stage from the moment of fixation) as compared with the control variants. The maximal sensitivity of the cells to the SCE induction by the mutagens is marked at the G1 stage of the first cell cycle before the moment of fixation. The level of SCE remains approximately the same in the second cell cycle before the moment of fixation (20-32 h) and decreased down to the control level at the G1 stage of the third cell cycle (48-52 h).     

Thiophosphamide induction of sister chromatid exchanges at various phases in the cell cycle of a Chinese hamster cell culture]

Influence of three concentrations of thiophosphamide (thioTEPA) on the formation of sister chromatid exchanges (SCE) has been studied at different phases during 2 cell cycles in cultured Chinese hamster cells. It is shown that the frequency of SCE does not differ from the control level under the effect of the mutagen on cells in the G2 phase of the first cell cycle from the moment of harvesting. Thiophosphamide induces the same number of SCE at S, G1 stages of the first cell cycle and G2 of the second one till the moment of harvesting. The number of SCE correlates in a direct proportion with a concentration of thiophosphamide. A scheme of forming SCE is proposed.     

In vivo sister chromatid differentiation and baseline sister chromatid exchanges in a mouse ascites tumour model.

Induction of differentially stained sister chromatids at G2/M and determination of baseline sister chromatid exchanges (SCEs) in ascites form of mouse sarcoma 180 cell line have been done by in vivo incorporation of 5-bromodeoxyuridine (BrdU) for two consecutive DNA replication cycles. The baseline SCE frequency is 6.24 at log phase of tumour growth.     

A decrease in sister chromatid exchange frequency during the prolonged cultivation of human lymphocytes

Sister chromatid exchange (SCE) frequency at different times of fixation was studied in human lymphocyte cultures obtained from 6 donors. No differences were found in the SCE frequency between human lymphocyte cultures fixed at 72 and 96 hours of incubation (10.61 +/- 0.85 and 10.15 +/- 0.81 SCE per cell, respectively). However, a decreased SCE frequency (8.11 +/- 0.36 SCE per cell) was observed in cultures fixed at 120 hours of incubation. For a more detailed studies, one lymphocyte culture was fixed at different times of incubation (from 56 to 128 hours, at each a 8 hours). A slight increase in SCE frequencies was found at the interval between 56 and 88 hours of incubation, while starting from 104 hours of incubation a marked decrease in the SCE frequency was observed. Time-dependent changes in the SCE frequency may be described by the equation y = -1.8614 + 0.3922x - (2.5183 x 10(-3))x2, where y is the number of SCEs per cell, and x--the duration of culture incubation in hours. The observed phenomenon may be associated with changes in proportion of T and B lymphocytes, or with heterochromatization of chromosomes during a prolonged cultivation, or with an early in vitro stimulation of the in vivo long-lived lymphocytes that may be more damaged than the in vivo short-lived and the in vitro late-stimulating ones.     

Statistical and image analysis of sister chromatid exchange in maize

The present study reports the use of the fluorescence plus Giemsa (FPG) technique, image analysis and statistical methods to assess the sister chromatid exchanges (SCEs) frequency in maize. Roots derived from germinated maize seeds were treated with BrdU solution and fixed. The slides were prepared by enzymatic cellular dissociation, air-drying technique, stained with Hoechst 33258 fluorochrome, and incubated in salt solution. The chromosomes were irradiated with ultraviolet light and stained with Giemsa solution. The FPG technique associated with digital analysis system was used to measure the length of 597 BrdU-incorporated maize chromosomes and to identify 0.5243 SCE per chromosome. A range from 0 to 4 SCE events were classified and the chi-square test (chi2=1.586, P=0.662) showed a good fit to the hypothesis that the SCEs are independent and random events that follow Poisson distribution. The SCE frequencies in long and short chromosome arms corresponded to a mean value of 0.876 SCE microm(-1). Considering that the maize line used in this study contains 5.78 picogram (pg) DNA (2C value) in interphasic G0/G1 nuclei or 11.56 pg DNA (4C value) in metaphase, and that the DNA mean value corresponds to 0.578 pg/metaphasic chromosome, the analysis suggests an occurrence of approximately 0.9 SCE/pg DNA.     

In vivo repair during G1 of DNA lesions eliciting sister chromatid exchanges induced by methylnitrosourea or ethylnitrosourea in BrdU substituted or unsubstituted DNA in murine salivary gland cells

The difference in efficiency of methylnitrosourea (MNU) and ethylnitrosourea (ENU) to induce SCE in early or late G1 was determined in synchronized murine salivary gland cells in vivo, as a measure of the capacity of this tissue to repair the lesions involved in SCE formation during G1. The repair during G1 was determined by treating the cells in early or late G1. Treatment was in the first cycle (G1 before incorporation of 5-bromodeoxyuridine (BrdU)) or in G1 of the second cycle (after a single round of BrdU incorporation). It was observed that 50% of the lesions induced by MNU that elicit SCE are repaired during G1. BrdU incorporation into DNA increases the sensitivity of the cell to SCE induction by MNU nearly 40%; however under this circumstance a slightly lower SCE frequency was observed in the cells exposed to MNU at early G1, indicating that during G1 only few lesions are repaired. The ENU-induced DNA-lesions involved in SCE production are nearly 100% persistent along G1; besides, a slight but significantly higher SCE frequency was observed in cells exposed at early G1, suggesting the formation of SCE-inducing lesions during G1. BrdU incorporation to DNA sensitizes the cell to SCE induction by ENU, increasing the SCE frequency to nearly to a 40%, although these additional lesions involved in SCE induction seem to be susceptible to repair during G1.     

Monitoring of premitotic and postmitotic apoptosis in gamma-irradiated HL-60 cells by the mitochondrial membrane protein-specific monoclonal antibody APO2.7

BACKGROUND: Majority of hematopoietic cells die by apoptosis after irradiation with ionizing radiation. In present study it is shown that human promyelocytic leukemia HL-60 cells can undergo two different types of apoptosis, premitotic and postmitotic. METHODS: HL-60 cells were irradiated with doses 8 and 20 Gy. For apoptosis detection APO2.7 antigen (mitochondrial membrane specific protein) expression without and with permeabilization by digitonin was used. This method was compared with flow-cytometric analysis of cell light scattering properties and determination of subG1 DNA. RESULT: Cells irradiated with high dose (20 Gy) died rapidly by premitotic apoptosis (interphase death) from all phases of cell cycle. 2 hours after irradiation cells with subdiploid DNA content and cells stained by APO2.7 after digitonin permeabilization appeared. After 6 hours 40% of cells were apoptotic, nonapoptotic cells were mainly in G1-phase. Lower dose (8 Gy) after 6 hours of irradiation caused accumulation of cells in S-phase. After 24 hours majority of cells was in G2-phase and apoptotic cells appeared (subG1 peak, APO2.7 with permeabilization). CONCLUSION: Data presented herein indicate that mitochondrial membrane protein-specific antibody APO2.7 after permeabilization is a useful marker for detection of early apoptotic cells dying by premitotic and postmitotic apoptosis.     

Analysis of bromodeoxyuridine-induced single and twin sister chromatid exchanges in tetraploid Chinese hamster ovary cells

Culture of cells in high exogenous levels (>10^–4 M) of bromodeoxyuridine (BrdUrd) or thymidine will increase the baseline sister chromatid exchange (SCE) frequency. The effect is thought to be related to the balance of the DNA precursors thymidine and deoxycytidine. Exogenous addition of deoxycytidine will reverse this effect. Single and twin SCEs were analysed in Colcemid-induced tetraploid Chinese hamster ovary cells exposed to different concentrations of BrdUrd to determine at what stage SCEs are induced by high levels of BrdUrd. In cells exposed to low concentrations of BrdUrd (10^–5 M), equal numbers of SCEs were induced in each of the two cell cycles. With increasing concentrations of BrdUrd (10^–4 to 2×10^–4 M), SCE frequency increased in both cell cycles, but far more SCEs were induced in the second cell cycle. Deoxycytidine (2×10^–4 M) reduced the frequency of SCEs primarily by reducing the frequency of SCEs induced in the second cell cycle. Treatment with 3-aminobenzamide (3AB), a potent inhibitor of poly(ADP-ribose) polymerase, produced effects similar to exposure to high levels of BrdUrd including inducing SCEs in the second replication cycle. This suggests a similar mechanism of action. Deoxycytidine had no effect on 3AB-induced SCEs, however, and there was no interaction between 3AB and high exogenous levels of BrdUrd in SCE induction. Thus these two agents probably act through different mechanisms.     

In vivo sister chromatid differentiation and base line sister chromatid exchanges in Channa punctatus.

An in vivo system for differentially stained sister chromatids by incorporating 5' Bromo 2' deoxyuridine at two consecutive round of DNA replication has been developed in C. punctatus. The base line developed frequency of sister chromatid exchanges (SCEs) was found to be 0.038 SCE/chromosome. This low baseline frequency of SCEs could be useful in detecting genotoxicity of pollutants in aquatic medium.     

Spontaneous level of sister chromosome exchanges and their distribution in human cells]

Blood of practically healthy donors of both sexes (27 females and 23 males) was cultured under the standard conditions during 96 hours. Bromodeoxyuridine (BUdR) was added at the final concentration of 10 mkg/ml 28 hours before harvesting. The slides were stained with acridine orange and Giemsa for differential staining of chromatids. In each culture sister chromatid exchanges (SCE) were analysed in 50 cells, and the part of cells undergoing the first, second and third mitoses at the time of harvesting, was calculated. According to the mean number of SCE per cell, the distribution of individuals was consistent with the normal law, the mean being 6.525 and standard deviation--0.956. A significant heterogeneity in the speed of cell cycle of cultures was observed. The coefficient of variation for the part of cells undergoing the first mitosis was 50%, for the cells in the second mitosis--15%, and for the cells in the third mitosis--154%. Correlation analysis showed a positive dependence of the mean level of SCF upon the age of a donor and upon the part of cells in the second mitosis in this individual. No reliable correlation of the SCE level with the donor's sex was observed. The distribution of cells, obtained from the culture of one individual, was best approximated by beta-distribution, and the distribution of cells obtained from the cultures of different individuals--by gamma-distribution. In both there was obtained a satisfactory approximation by Pearson's distribution of the 1 type, and significant deviations were found from the normal, Poison's and the negative binomial distribution. The conditions were found of similarity of empirical distribution of SCE in cells to the normal one. For that, it is not the value of SCE for a separate cell that should be used as a unit of measurement, but the mean from the values of frequencies for 5-10 cells. Hence, it was shown that for the evaluation of the mean frequency of SCE with the precision of 1 exchange in separate individuals it is necessary to analyse 40 cells, and to observe the 15% increase of spontaneous SCE level under the action of deleterious factors--8 individuals are enough to analyse.     

Three-way differential staining of sister chromatids in M3 chromosomes : Evidence for spontaneous sister chromatid exchanges in vitro

Three types of Giemsa differential staining of sister chromatids were observed in HeLa cells when they were exposed continuously to 5-bromodeoxyuridine (BrdUrd) for three replication cycles. In type-1, about a half set of chromosome complements were composed of pairs of darkly-stained and intermediately-stained chromatids; the other half consisted of pairs of intermediately-stained and lightly-stained chromatids. In type-2, one fourth of chromatids was stained darkly and the remaining ones were stained lightly. In type-3, about a half set of chromosomes consisted of the pairs of darkly-stained and lightly-stained chromatids and the rest of pairs of intermediately-stained and lightly-stained chromatids. Cells showing each differentiation pattern at the third mitotic phase were dependent on the stages of the first DNA synthetic (S) phase at which BrdUrd treatments were initiated. Type-1 cells were observed, when BrdUrd treatment was initiated anywhere from G1 to early S phase, type-2 when treatments were begun in middle S stage, and type-3 when treatments were initiated in the late stages of the first S phase. The appearance of the three types seems to be caused by a different amount of BrdUrd incorporated into DNA between the first (S1) and the second S period (S2). The amount of BrdUrd incorporated is as follows: in type-1 S1>S2, in type-2 S1 S2 and in type-3 S2>S1.By analysing type-1 cells, all of the sister chromatid exchanges (SCEs) occurring during each replication cycle can be accurately counted and distinguished from one another. In cells exposed to BrdUrd above 5 μg/ml, the frequencies of SCEs occurring during S1, S2, and S3 are higher than those detected at lower BrdUrd concentrations. On the other hand, at lower concentrations (0.1–1.0 μg/ml) they occurred at the same frequency during S1, S2, and S3. Thus, SCEs detected at low concentrations are free from the incremental effect of BrdUrd incorporated, and enable us to estimate the spontaneous level of SCE frequency.     

The influence of G2 progression on X-ray sensitivity of two-cell mouse embryos

Naturally synchronous, two-cell mouse embryos were X-irradiated in vitro. In experiment 1, irradiation was either in the early or in the late G2 phase, which lasts about 14 hours. In experiment 2, irradiation of all the embryos was in late G2 but embryos with different intervals between irradiation and the first mitosis after irradiation were separated and investigated independently. After 2 Gy the time interval between irradiation in late G2 and the first mitosis post-irradiation was on the average about 9 hours; after irradiation in the early G2 phase about 13.5 hours. Development (hatching of blastocysts) and cell proliferation (cell number per embryo at the stage of the hatched blastocyst) was most impaired and the frequency of micronuclei (determined in four- or eight-cell embryos) was highest in the case of a short interval between irradiation in G2 and the first mitosis post-irradiation. It is concluded that a longer interval allows a longer period of DNA repair. The results also demonstrate a positive correlation between the extent of chromosomal damage (micronuclei) and the extent of cell death as well as the impairment of the development of the whole biological system.     

Effect of the time before fixation on the yield of chromosome aberrations in a culture of human lymphocytes exposed to gamma rays at different stages of the mitotic cycle

A comparative study was made of the yield of chromosome aberrations in human lymphocyte culture exposed to 60Co-gamma-rays (2 Gy) at different mitotic cycle stages the cells being fixed after 52 and 60 hr. It was shown that with the latter fixation time (60 hr) the frequency of chromosome aberrations after irradiation in G1 stage was substantially lower than that with the former one (52 hr) and, vice versa, it was higher after irradiation in S and G2 stages. The authors discuss the probable causes of the distinctions observed.     

Distribution of sister chromatid exchanges in the euchromatin and heterochromatin of the Indian muntjac

The frequency of sister chromatid exchanges (SCEs) was determined for the chromosomes (except Y2) of the Indian muntjac stained by the fluorescence plus Giemsa (FPG) or harlequin chromosome technique. The relative DNA content of each of the chromosomes was also measured by scanning cytophotometry. After growth in bromodeoxyuridine (BrdU) for two DNA replication cycles. SCEs were distributed according to the Poisson formula in each of the chromosomes. The frequency of SCE in each of the chromosomes was directly proportional to DNA content. A more detailed analysis of SCEs was performed for the three morphologically distinguishable regions of the X-autosome composite chromosome. The SCE frequency in the euchromatic long arm and short arm were proportional to the amount of DNA. In contrast, the constitutive heterochromatin in the neck of this chromosome contained far fewer SCEs than expected on the basis of the amount of DNA in this region. A high frequency of SCE, however, was observed at the point junctions between the euchromatin and heterochromatin.     

A change in sister chromatid behavior precedes nuclear division in Saccharomyces cerevisiae

We used a genetic assay to monitor the behavior of sister chromatids during the cell cycle. We show that the ability to induce sister chromatid exchanges (SCE) with ionizing radiation is maximal in budded cells with undivided nuclei and then decreases prior to nuclear division. SCE can be induced in cells arrested in G2 using either nocodazole or cdc mutants. These data show that sister chromatids have two different states prior to nuclear division. We suggest that the sister chromatids of cir. III, a circular derivative of chromosome III, separate (anaphase A) prior to spindle elongation (anaphase B). Other interpretations are also discussed. SCE can be induced in cdc mutants that arrest in G2 and in nocodazole-treated cells, suggesting that mitotic checkpoints arrest cells prior to sister chromatid separation.     

Effect of free 5-bromodeoxyuridine on induction of SCE in human lymphocytes gamma-irradiated at the presynthetic stage of primary and secondary mitotic cycles

Formation of SCE was studied in lymphocytes irradiated by 60Co gamma-rays at the G1 stage of the first or second mitotic cycles. The yield of SCEs induced by irradiation in the presence of 5-bromodeoxyuridine (BrdU) proved to be significantly higher than that obtained in the absence of BrdU. The enhancing influence of BrdU on SCE induction depends on neither replication cycle nor the molecular constitution of chromosomes under irradiation. Direct modification of chromosomal radiosensitivity by BrdU is excluded. The results obtained suggest the interference of free BrdU present in culture medium with processes of DNA reparation at the G1 stage.     

Dynamics of the frequencies of chemically induced sister chromatid exchanges in a series of cell generations

The number of sister chromatid exchanges (SCE) in a culture of human lymphocytes and the established cultures of Chinese hamster cells has been studied after the exposure to thiophosphamide and dipin at different stages of the cell cycle. The most pronounced effect is observed under the action of the mutagens at the G1 of the first cycle, prior to harvest. The SCE level becomes lower with the increase of the interval between the exposure to the mutagen and the time of 5-BUdR introduction. The number of SCEs per cell drops to the control level when the duration of the first interval is equal to that of the cell cycle. The results obtained prove the mutagen-induced impairments causing SCE formation to be repaired practically 100%, provided that at least one cycle of DNA replication took place.     

Effect of 5-bromodeoxyuridine substitution on sister chromatid exchange induction by chemicals

The fluorescence-plus-Giemsa (FPG) technique for analysis of sister chromatid exchange (SCE) is widely used as an assay for mutagenic carcinogens. There is very little information, however, on whether incorporation of the bromodeoxyuridine (BrdU) necessary for visualization of SCEs affects the sensitivity of the SCE test system to different chemical agents. We have investigated the effect of BrdU incorporation on SCE induction by labeling cells with BrdU for either the first cell cycle or the first and second cell cycles. The cells were then treated with bleomycin, which produces DNA strand breakage; proflavine, which intercalates into DNA; mitomycin C, which produces monoadducts and DNA crosslinks; or aphidicolin, which inhibits DNA polymerase . Chemicals were added before BrdU exposure or during the first, second, or both cell cycles. Only mitomycin C, which induces long-lived lesions, elevated the SCE frequency when cells were treated before BrdU labeling. When bleomycin, proflavine, or mitomycin C was present concurrently with BrdU, the frequency of SCEs was increased independently of the BrdU labeling protocol. Aphidicolin, on the other hand, induced more SCEs when present for the second cell cycle, when DNA replicates on a template DNA strand containing BrdU. We also examined the induction of SCEs in the first cell cycle (twins) and in the second cell cycle (singles) after continuous treatment of cells with BrdU and the test chemicals. Only aphidicolin increased SCE frequency in the second cell cycle. These results indicate that aphidicolin, but not bleomycin, proflavine, or mitomycin C, affects BrdU-substituted DNA and unsubstituted DNA differently. This type of interaction should be taken into consideration when the SCE test is used as an assay system.     

In vivo sister chromatid exchange in cells of various organs of the mouse

In vivo sister chromatid exchange (SCE) in mouse cells derived from various organs was studied by infusing BrdU from the tail vein. It was found that at BrdU concentrations ranging from 2.2–13.5 g/g/h, the SCE frequency in bone marrow cells seemed to stay at a constant level (1.5–2/cell/two cell cycles) whereas it started to rise as the BrdU dose exceeded this dose range. When BrdU within this dose range was infused continuously from the tail vein for appropriate hours to label chromosomes in various organs, the average SCE frequencies per cell were found to be 1.64 in bone marrow cells, 1.82 in spermatogonia, 1.99 in splenic cells, 2.89 in intestinal cells and 3.69 in cells from adjuvant stimulated lymph nodes. It is suggested that the spontaneous level of the in vivo SCE frequency might be about 1.5–2/cell/two cell cycles in the mouse. In cells derived from intestine and adjuvant stimulated lymph node, some unknown factors might work as a inducer of SCEs resulting in a significant increase in the SCE frequency in these organs.     

A change in sister chromatid behavior precedes nuclear division in Saccharomyces cerevisiae

We used a genetic assay to monitor the behavior of sister chromatids during the cell cycle. We show that the ability to induce sister chromatid exchanges (SCE) with ionizing radiation is maximal in budded cells with undivided nuclei and then decreases prior to nuclear division. SCE can be induced in cells arrested in G2 using either nocodazole or cdc mutants. These data show that sister chromatids have two different states prior to nuclear division. We suggest that the sister chromatids of cir. III, a circular derivative of chromosome III, separate (anaphase A) prior to spindle elongation (anaphase B). Other interpretations are also discussed. SCE can be induced in cdc mutants that arrest in G2 and in nocodazole-treated cells, suggesting that mitotic checkpoints arrest cells prior to sister chromatid separation. Received: 3 July 1996 / Accepted: 4 October 1996