Analysis of baseline and BrdU-dependent SCEs at different BrdU concentrations

In the present paper we have used a rationale based on the development of theoretical equations that define sister-chromatid exchange (SCE) frequencies as a function of two variables, namely the baseline (BrdU-independent) and the BrdU-dependent SCE frequencies. The experimental design includes the estimation of SCE frequencies in second division chromosomes when both cycles occurred in the presence of BrdU and when BrdU incubation took place only during the first cycle in a wide range of BrdU concentrations. The final SCE yields in second division chromosomes could be separated into three different components: (1) The BrdU-independent, ‘spontaneous’ or baseline SCEs, whose low but biologically significant frequency was calculated to be about 0.06 SCEs per pg of DNA; this figure could be similar for most of the cell types; (2) the BrdU-dependent SCEs whose frequency increases with BrdU dose, probably as a result of BrdU substitution for thymidine; (3) the BrdU-dependent SCEs as a consequence of other cellular factors such as disturbance of nucleotide pool sizes. At high BrdU concentrations (300 μM upward) the three components appear to have a significant value in the final SCE yield, whereas at lower BrdU doses the third component seems to be negligible.     

SCEs are induced by replication of BrdU-substituted DNA templates, but not by incorporation of BrdU into nascent DNA

After 3 rounds of DNA replication in the presence of BrdU, third-division metaphase cells can be scored for the frequencies of SCEs that occurred during cycles 1 and 2, and also for the frequency of SCE during cycle 3. This procedure was used to resolve the issue of SCE induction by replication of BrdU-substituted DNA templates versus induction by BrdU incorporation into nascent DNA. It was observed that third-cycle SCE frequencies in CHO are dependent upon the amount of BrdU that was present during cycles 1 and 2 and are independent of the BrdU concentration during the third cycle. It is therefore BrdU serving as a template, rather than BrdU being incorporated, that initiates the SCE event. A model is proposed that produces reasonable fits to the observed data. It also predicts a true background or spontaneous SCE frequency of 3 per cell per cycle as previously reported by Heartlein et al. (Mutation Res., 107 (1983) (103-109). The predicted single twin ratio is higher than that reported by Wolff and Perry (Exp. Cell Res., 93 (1975) 23-30), and possible explanations for this discrepancy are discussed.     

Frequency of sister-chromatid exchanges depending on the amount of 5-bromodeoxyuridine incorporated into parental DNA

Chinese hamster D-6 cells were grown for two cell cycles. The effect of 5-bromodeoxyuridine (BrdU) on the frequencies of sister-chromatid exchanges (SCEs) in these cells was investigated by the BrdU-labeling method. A low concentration (5 μM) of BrdU was inoculated in the first cell cycle for SCE counting. When excess concentrations (100–1000 μM) of BrdU were added subsequently in the second cell cycle, a 1–2-fold increase of SCE frequencies was observed. When excess thymidine (dT) (100–1000 μM) was supplied instead of BrdU, the incidence of SCE also increased. When cells were exposed to high concentrations (50–200 μM) of BrdU in the first cell cycle, a 1–4-fold increase in SCE frequencies was observed. This incidence of SCE was largely dependent on the concentration of BrdU and dT used in the second cell cycle. These results suggest that efficient SCE induction by BrdU is related to the BrdU residue incorporated into parental DNA strands.     

Induction of endoreduplication by hydrazine in Chinese hamster V 79 cells and reduced incidence of sister chromatid exchanges in endoreduplicated mitoses

Hydrazine in high concentrations very effectively induces endoreduplication in Chinese hamster V 79 cells. The addition of 5-bromodeoxyuridine (BrdU) for the duration of one cell cycle prior to the induction of endoreduplication produces diplochromosomes with sister chromatid differentiation (SCD) after differential chromatid staining. The fact that diplochromosomes with complete SCD are obtained shows that endoreduplication was induced in cells that were in G2-phase. The analysis of sister chromatid exchanges (SCEs) showed that hydrazine treatment rarely led to increased SCE frequencies in mitoses after endoreduplication, but that it caused a strong SCE induction in diploid second division metaphases in the same culture. Neither catalase nor cysteine had an effect on the induction of endoreduplication or the incidence of SCEs. Treatment of the cells with mitomycin C prior to addition of BrdU led to increased SCE frequencies. Compared with the normal mitoses from the same preparation, the mitoses after endoreduplication showed a significantly reduced induction of SCEs. In contrast to these findings, SCE induction was not reduced in the common tetraploid V 79 cells after colcemid-induced polyploidization.     

Increased sister-chromatid exchanges (SCEs) and chromosomal fragilities by BrdU in a human mutant B-lymphoblastoid cell line

From an X-irradiated human B-lymphoblastoid cell line (CCRF-SB), we have isolated a unique mutant clone (CCRF-SB-T1) which reveals high frequencies of sister-chromatid exchanges (SCEs) and chromosomal fragilities in the C-band regions of chromosomes Nos. 1, 9 and 16, when exposed to high concentrations of bromodeoxyuridine (BrdU). A clear BrdU dose-dependent increase of SCEs (9.6 SCEs/cell at 0.05 mM, 40 SCEs/cell at 0.37 mM on average) in this mutant was observed. Relative contributions of nucleoside and a thymidine (dT) analog of BrdU to high SCEs were studied, since an unusual SCE response to BrdU led us to suspect the significance of BrdU incorporation into DNA and dT pool disturbances. Addition of deoxycytidine (dC), dT or both dC and dT causes an increase of SCEs. On the other hand, deoxyadenosine (dA) and deoxyguanosine (dG) did not have significant effects on SCEs in SB-T1 cells. These results suggest that disturbances of pyrimidine-nucleotide synthesis, including gross imbalance of nucleotide pools, play a pivotal role in the high SCE induction of SB-T1 cells by BrdU.     

Differential fluorescence of sister chromatids in chicken embryos exposed to 5-bromodeoxyuridine

Differential fluorescence of sister chromatids (SCD) and sister chromatid exchanges (SCE) were visualized in chromosomes obtained directly from growing chicken embryos. SCD was obtained by exposing 3-day embryos to BrdU (12.5-50 mug) in ovo for 26 hours and staining air dried chromosome preparations with 33258 Hoechst. Bright, stable fluorescence and continued SCD were achieved if slides were mounted in McIlvaine's pH 4.4 buffer. Embryo growth, mitotic activity and gross chromosome morphology were not adversely altered by the BrdU treatments. The SCE rate was estimated to be 0.07 SCEs per macrochromosome and 0.75 SCEs per metaphase for two cell cycles.     

SCE levels in Bloom-syndrome cells at very low bromodeoxyuridine (BrdU) concentrations: monoclonal anti-BrdU antibody

A stable staining procedure of sister-chromatid differentiation (SCD) using a monoclonal antibromodeoxyuridine (BrdU) antibody was newly established by combining it with the immunoperoxidase reaction (3,3'-diaminobenzidine, DAB reaction). This procedure permitted detection of SCD and SCE at very low BrdU concentrations. SCD was not usually observed below 2.0 micrograms/ml BrdU with flame-dried chromosome slides. When chromosome slides were prepared by air-drying over 37 degrees C warm water, SCD was detected at 10.0, 5.0, 1.0, 0.5, 0.3 and 0.2 micrograms/ml BrdU with FPG and even at 0.1 microgram/ml BrdU with the antibody technique. SCE levels were evaluated using the antibody technique and endomitotic analysis with FPG at low BrdU concentrations (1.0, 0.5, 0.3, 0.2 microgram/ml) in two BS B-lymphoblastoid cell lines (LCLs). Even though the BS SCE level was approximately 70 per cell at 10 micrograms/ml, the value decreased to the level of 20-30 SCE per cell at 0.1 microgram/ml with the antibody technique. In BrdU-labelled BS endomitoses, single SCEs highly decreased with BrdU concentrations (130-140 level at 10 micrograms/ml: 38-60 level at 0.2 microgram/ml), when compared to the rare twin SCE values (3-6 SCE level) at all BrdU concentrations. These findings conclusively indicate that the spontaneous baseline SCE in BS B-lymphoblastoid cells is low and most BS SCEs are caused by BrdU.     

A unique human mutant B-lymphoblastoid cell line (ataxia telangiectasia) which exhibits increased siste-chromatid exchange retaining hypersensitivity to neocarzinostatin and bleomycin

Chromosome aberrations and sister-chromatid exchanges (SCEs) were examined in 4 ataxia telangiectasia (AT)-derived B-lymphoblastoid cell lines (B-LCLs) (AT-S, AT-SHI, AT-SHI B13A and AsHa) following treatments with neocarzinostatin (NCS) and bleomycin. All of these cell lines exhibited extremely high frequencies of chromosome aberrations with the NCS and bleomycin treatments. Among them, AsHa, a mutant B-LCL originating from an AT patient, showed high frequencies of SCEs under high bromodeoxyuridine (BrdU) concentrations retaining hypersensitivity to NCS and bleomycin with regard to chromosome aberrations. A clear BrdU dose-dependent increase in SCEs (9.85 SCEs/cell at 40 μg/ml, 36.65 SCEs/cell at 100 μg/ml on average) in this mutant was observed. When AsHa mutant cells were treated with NCS (0.02 μg/ml) and/or bleomycin (5.0 μg/ml) under 40 μg/ml BrdU (minimum BrdU concentration for sister-chromatid differential staining), SCE levels increased from 9.85 (baseline level) to 21.1 with NCS and 20.5 with bleomycin, in a dose-dependent manner. These observations indicate that AsHa is a unique AT-derived mutant cell clone with a high SCE character retaining the original hypersensitivity to bleomycin and NCS.     

Distribution of sister chromatid exchanges on the mouse chromosomes in vivo with reference to the replication properties of the X chromosome

Frequency of sister chromatid exchanges (SCE) were recorded separately for different chromosomes from bone marrow cells of female mice of the two genetic strains (C3H/S and C57BL/6J). SCEs were evaluated following different doses of 5-bromo-2'-deoxyuridine (BrdU) as nine hourly i.p. injections. The SCE per cell increased with increasing BrdU doses which was slightly higher in C3H/S than in the C57BL/6J. SCEs per cell were variable at every treatment-strain combination, possibly reflecting the heterogeneous nature of the bone marrow cells. In general, there is a positive correlation between SCE per chromosome and the relative chromosome length. Total SCEs on one of the large chromosomes (most likely the X chromosome), however, are significantly higher than expected on the basis of relative length alone. Most of this increase is attributable to one of the homologues of this chromosome, which is not in synchrony with the rest of the chromosomes and may represent the late-replicating X. These results when viewed in the light of replication properties of the heterochromatinized X, suggest a direct involvement of DNA replication in SCE formation and may argue against the replication point as the sole site for the SCEs.     

Influence of low doses of BrdU and estimation of spontaneous SCE in CHO chromosomes: three-way differential staining and an immunoperoxidase method

The influence of low doses of 5-bromodeoxyuridine (BrdU) on the occurrence of sister chromatid exchanges (SCEs) during the first cell cycle, when unsubstituted DNA templates replicate in the presence of the halogenated nucleoside (SCE1) has been assessed in third mitosis (M3) Chinese hamster ovary (CHO) cells showing three-way differential (TWD) staining. In addition, lower concentrations of BrdU, not detectable by Giemsa staining, have been tested by a high resolution immunoperoxidase method (anti-BrdU monoclonal antibody) and SCEs were scored in second mitosis (M2) cells. Our findings was a dose-response curve for SCE1 that allows an estimated mean spontaneous yield of 1.32/cell per cell cycle by extrapolation to zero concentration of BrdU. On the other hand, when the total SCE frequency corresponding to the first and second rounds of replication (SCE1+SCE2) found in M3 chromosomes was compared with the yield of SCEs scored in M2 cells grown in BrdU at doses lower than 1 M no further reduction was achieved. This seems to indicate that SCEs can occur spontaneously in this cell line, though the estimated frequency is higher than that reported in vivo.by S. Wolff     

Spontaneous sister-chromatid exchange frequencies in human B and T lymphocytes at BrdU borderline concentrations for sister-chromatid differentiation

Human peripheral blood B and T lymphocytes, highly purified by immunologic methods, were supplemented with gamma-irradiated unseparated autologous mononuclear cells to restore helper functions and stimulated with pokeweed mitogen and phytohemagglutinin, respectively. Spontaneous sister-chromatid exchange (SCE) frequencies were investigated in proliferating B and T lymphocyte cultures labeled with the cell-type-specific borderline concentrations of 5-bromodeoxyuridine (BrdU) for sister-chromatid differentiation (SCD). B lymphocytes from 6 different donors showed mean values of 3.28-3.72 SCE events/cell. In T lymphocytes, mean values of 6.30-7.28 SCEs/cell were observed. The differences between the SCE distributions of the cell populations are highly significant. The results show that the differences in the spontaneous SCE frequencies between human B and T lymphocytes were not due to a difference in the uptake of BrdU.     

Presence of a Base Line Level of Sister Chromatid Exchanges in Somatic Cells of DROSOPHILA MELANOGASTER IN VIVO and IN VITRO

Sister chromatid exchanges (SCEs) under in vivo and in vitro conditions were examined in ganglion cells of third-instar larvae of Drosophila melanogaster (Oregon-R). In the in vivo experiment, third-instar larvae were fed on synthetic media containing 5-bromo-2'-deoxyuridine (BrdUrd). After two cell cycles, ganglia were dissected and treated with colchicine. In the in vitro experiment, the ganglia were also incubated in media containing BrdUrd for two cell cycles, and treated with colchicine. SCEs were scored in metaphase stained with Hoechst 33258 plus Giemsa. The frequencies of SCEs stayed constant in the range of 25-150 micrograms/ml and 0.25-2.5 micrograms/ml of BrdUrd in vivo and in vitro, respectively. SCEs gradually increased at higher concentrations, strongly suggesting that at least a fraction of the detected SCEs are spontaneous. The constant levels of SCE frequency were estimated, on the average, at 0.103 per cell per two cell cycles for females and 0.101 for males in vivo and at 0.096 for females and 0.091 for males in vitro. No difference was found in the SCE frequency between sexes at any of the BrdUrd concentrations. The analysis for the distribution of SCEs within chromosomes revealed an extraordinarily high proportion of the SCEs at the junctions between euchromatin and heterochromatin; the remaining SCEs were preferentially localized in the euchromatic regions of the chromosomes and in the heterochromatic Y chromosome. These results were largely inconsistent with those of Gatti et al. (1979).     

Comparisons of in vivo BrdU labeling methods and spontaneous sister chromatid exchange frequencies in regenerating murine liver and bone marrow cells

BrdU and BrdC have been employed as DNA labeling agents for differentiation of sister chromatids and for extension of sister chromatid exchange (SCE) methods to regenerating murine liver cells in vivo. Comparisons were made between bone marrow and liver cells isolated simultaneously from mice following DNA labeling with either BrdC or BrdU. Although the total mitotic yield of bone marrow cells was considerably greater than in liver, a higher percentage of second division metaphases was observed in liver cell preparations. The percentages of second division c-metaphase cells observed were 31.5% in bone marrow and 73% in liver cell preparations. Utilizing either BrdU or BrdC, no significant difference in percentage of second division metaphases was discerned. The number of spontaneous SCEs per cell was distributed according to the Poisson probability function. No significant differences in mean numbers of SCEs per cell were found in comparisons of bone marrow (1.40) and liver cells (1.65) or of cells which had incorporated BrdU or BrdC.     

Effects of cell fusion and deoxynucleosides on sister-chromatid exchanges in B-lymphoblastoid cell lines from 5 Bloom syndrome patients

The effect of cell fusion and deoxynucleosides (deoxyadenosine, dA; deoxyguanosine, dG; deoxycytidine, dC; thymidine, T) on sister-chromatid exchanges (SCEs) in Bloom syndrome (BS) was studied in two types of BrdU (bromodeoxyuridine)-sensitive and BrdU-resistant B-lymphoblastoid cell lines (LCLs) with respect to cellular proliferation in BrdU-labeled culture conditions. Cell fusion between BrdU-sensitive and BrdU-resistant BS B-LCLs did not exhibit complementation, although when any of the BS B-LCLs (retaining high SCE character) labeled with BrdU were fused with non-labeled normal cells, the hybrid cells had a normal level of SCE at the first mitosis after fusion. Deoxycytidine addition showed no effect on SCEs in normal cells but decreased SCEs in BS cells from the baseline level of 70 SCEs/cell to about 60 SCE/cell. Purine deoxyribonucleosides (dG and dA) caused a significant concentration-dependent increase in SCE frequency both in normal and BS cells. Although T caused a 2-fold increase in normal SCEs, it highly decreased BS SCE from 70 SCEs/cell to 35 SCEs/cell. FrdU did not greatly affect BS SCE in the presence of BrdU and T. These observations indicate strongly that BS cells may have a low thymidine pool compared with normal cells, which could account for a more efficient BrdU substitution in the DNA thus potentiating the template effect on SCE.     

Do the frequencies of sister chromatid exchanges in endoreduplieated mitoses provide a measure for lesion persistence and repair?

Endoreduplication was induced in V 79 cells using Colcemid. The concentration of Colcemid necessary to induce endoreduplication is about 1000 times higher than that needed to arrest mitoses or to induce ordinary tetraploid cells. Diplochromosomes with sister chromatid differentiation were obtained by adding BrdU for the duration of one cell cycle prior to the induction of endoreduplication. The induction of endoreduplication with Colcemid had no influence on the frequency of sister chromatid exchanges (SCEs). Treating the cultures with mitomycin C (MMC) before adding BrdU increased the percentage of endoreduplieated mitoses and also led to marked SCE induction. In the diplochromosomes, the frequencies of both twin SCEs (first cycle) as well as single SCEs (second cycle) were increased. It was also found that the SCE frequencies in mitoses after endoreduplication were lower than the values found in diploid and ordinary tetraploid metaphases of the same preparation. The possible conclusions concerning the lifetime of SCE-inducing lesions and the influence of repair processes are discussed.     

Bromodeoxyuridine (BrdU) template and thymodine pool effects on high frequencies of sister-chromatid exchange (SCE) in Bloom syndrome cells and a mutant cell line (AsHa) originated from ataxia telangiectasia

The effect of bromodeoxyuridine (BrdU)-substituted DNA template and thymidine (dT) pool on excess sister-chromatid exchanges (SCEs) was studied in Bloom syndrome (BS) cells and an ataxia telangiectasia (AT)-derived mutant cell line (AsHa). When BS endomitotic cells were labeled with low and high (or high and low) BrdU concentrations during S₁ and S₂, only the BrdU concentration during S₁ phase affected the observed SCE. In BS cells about a 10-fold increase in SCEs occurs during or following replication on a BrdU-substituted template (high-high and high-low BrdU labeling) relative to the normal DNA template. SCEs decreased to about half in AsHa cells labeled with various BrdU doses (40, 60, 80 and 100 μg/ml) during only S₁, compared with those labeled during S₁ and S₂. Co-cultivation of AsHa and BS cells resulted in a significant reduction in SCE level from 70 to 13–17 in BS cells, lowered the BrdU concentrations necessary for sister-chromatid differential (SCD) staining from 40 to 10 μg/ml with normal SCE level and resulted in decreased level of SCEs at high BrdU concentrations (80–100 μg/ml) 12–14 SCE) in AsHa cells, compared with the originally increased SCE level (36.65 SCE at 100 μg/ml) without co-culture. However, co-cultivation between AsHa and normal cells lowered the BrdU dose necessary for SCD staining from 40 to 30 μg/ml; the dT pool possibly balanced at this level, which is clearly higher than that at co-cultivation between AsHa and BS cells. The reason for the very high BrdU doses needed to achieve SCD would seem to be that AsHa cells have high levels of thymidylate (TMP) synthetase, which maintain a large endogenous thymidine pool. This has been confirmed by direct measurement. These findings strongly support that excess and decreased dT pools are closely related to the condition necessary for high SCE induction.     

In vitro SCE discrepancy between embryonic and extraembryonic mouse tissues

In vitro sister-chromatid exchange (SCE) background levels and cytokinetics were compared in embryonic (whole embryo cell suspensions) and extraembryonic (yolk sac and amnion, placenta) cells of inbred and outbred strains at various gestational stages (days 12-17). Results indicate a tissue origin (embryonal, extraembryonal) related variation in the formation of baseline SCE frequencies and cytokinetics. The significant higher SCE levels in extraembryonic tissues (maximum increase of 2 X the background values of the embryo cells) were independent of mouse strain and gestational stage. An average of 4-5 SCEs/cell in embryo cells is contrasted by 7-9 SCEs/cell in extraembryo cells. Mitotic index was generally lower and average generation time longer (by 2-3 h) in extraembryonic tissue cells. No significant differences in SCE frequencies and no changes in cytokinetics were detected at the BrdU concentrations used (1.2-4.8 micrograms/ml). The reason for the inter-tissue differences in baseline SCE is still not clear.     

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.     

Increased sister chromatid exchange events in the human late replicating X

Summary Human female blood cultures were labeled with BrdU for detecting sister chromatid exchanges (SCEs) by the Hoechst 33258 fluorescence technique. Late labeling with ³H-thymidine and autoradiography allowed the identification of the late replicating X. The mean number of SCEs in the cells was 13. The isopycnotic X showed an exchange frequency according to its relative length in the karyotype; in the late replicating X a doubled number of SCE events was observed.     

Determination of the baseline sister chromatid exchange frequency in human and mouse peripheral lymphocytes using monoclonal antibodies and very low doses of bromodeoxyuridine

We measured the frequency of sister chromatid exchanges (SCEs) in human and mouse peripheral lymphocytes using doses of bromodeoxyuridine (BrdU) ranging from 30 nM to 100 microM (human) and from 10 nM to 10 microM (mouse). Heparinized peripheral blood was obtained from five healthy nonsmokers and from six C57B1/6 male mice. The blood was stimulated with PHA (human) or lipopolysaccharide (LPS, mouse) and grown for the first of two cell cycles in BrdU. Metaphase chromosomes were denatured and exposed to a monoclonal antibody reactive to single-stranded DNA containing BrdU. A second antibody was used to label the first antibody with fluorescein, and propidium iodide was used as a counterstain. Second-division metaphases were thus differentially stained red to indicate DNA content and yellow-green to indicate the presence of BrdU. The results indicate that the baseline SCE frequency in human and mouse peripheral lymphocytes is 3.6 and 2.4 SCEs per cell per generation, and that in the human these frequencies are invariant at the lowest BrdU levels. This suggests that SCEs are an integral part of DNA replication, even in the absence of agents known to induce SCEs. The distribution of SCEs per chromosome was analyzed and found to be Poisson-distributed in all 24 murine cultures and in 25 of 36 human cultures. The distribution of SCEs per chromosome may be due to either species-specific chromosome packaging or to karyotypic differences between the species.