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.     

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.     

Demonstration of sister chromatid differentiation in human amniotic fluid cells after partial synchronization with BrdU or dT surplus

Summary Protocols are compared demonstrating sister chromatid differentiation (SCD) in human amniotic fluid (AF) cells with and without partial synchronization. Partial synchronization both with an excess of 5-bromodeoxyuridine (BrdU) and an excess of thymidine leads to an increase of metaphases with SCD. Compared with unsynchronized cells, the rate of sister chromatid exchanges (SCE) is not increased. Studies on the late replicating X chromosome of female cells showed that the addition of mitomycin C (MMC) after releasing the thymidine block preferentially induces SCEs in late replicating regions. The partial synchronization with thymidine surplus provides a good basis for SCE experiments with AF cells and facilitates the prenatal diagnosis of diseases characterized by changes in the SCE rate.     

Analysis of low concentrations of 5-bromo-2-deoxyuridine on sister chromatid exchanges in human lymphocytes

To determine a concentration of 5-bromo-2-deoxyuridine (BrdU) sufficient for sister chromatid differentiation (SCD), and yet having a minimal effect on the number of sister chromatid exchanges (SCEs), we assessed the effect produced on the number of SCEs by low concentrations (1, 3, and 10 micrograms/mL) of BrdU. SCD was not obtained in 19% of the 31 subjects with 1 microgram/mL of BrdU, while the differentiation was adequate for all samples treated with 3 and 10 micrograms/mL. We statistically analysed the effects of these three different doses and found no significant difference in the number of SCEs obtained with the doses of 1 and 3 micrograms/mL, but a significant difference was observed between these two concentrations and 10 micrograms/mL. We therefore suggest that the dose of 3 micrograms/mL, while sufficient to produce reliable differential staining, still permits an adequate evaluation of the base line of SCEs and appears to enhance the sensitivity of the test to evaluate between-individual variations. Our experiments also underline that SCE counts should include the centromere exchanges.     

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.     

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.     

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.     

Sister chromatid differentiation and chromosomal in situ suppression hybridization: a combined methodology for analyzing cell proliferation and SCEs in individual chromosomes.

A technique combining sister chromatid differentiation (SCD) with chromosomal in situ suppression (CISS) hybridization is described. This combined methodology allows simultaneous analysis of cell-proliferation kinetics and sister chromatid exchanges (SCEs) in chromosomes identified by probes. To demonstrate the usefulness of this approach, cultured fibroblasts from a patient with Pallister-Killian syndrome, mos46/47,+i(12p), a chromosome mosaicism disorder, were studied. The fibroblasts were cultured in the presence of 5-bromodeoxyuridine (BrdU) for 72 h. Chromosome preparations were stained by a modified fluorescence-plus-Giemsa method to obtain SCD. For identification of the normal chromosome 12 and the i(12p), CISS hybridization with a biotin-labeled chromosome 12-specific library probe (LA 12NS01) was carried out after SCD. The hybridization was detected by an indirect immunofluorescence technique. For the analysis of cell kinetics and SCEs, the technique allows rapid, reliable identification of abnormal and normal cell populations. It also allows analysis of SCEs in individual chromosomes.     

Three-way differentiation of sister chromatids in endoreduplicated (M3) chromosomes of Bloom syndrome B-lymphoid cell line

Summary The three-way differentiation of sister chromatids (3-way SCD) in M₃ endoreduplicated chromosomes in a Bloom syndrome (BS) B-lymphoid cell line, suggested that in addition to exchanges between sister chromatids (intra-exchanges), non-sister chromatid exchanges (inter-exchanges) also occur, especially in BS high SCE cells. In BS diploid chromosomes such inter-exchanges probably get confused with intra-exchanges when total SCEs are accounted for. Bloom syndrome high SCE cells probably do not follow the same bromodeoxyuridine (BrdU) uptake pattern over three cell cycles as normal cells. The 3-way SCD in M₃ endoreduplicated chromosomes can be explained on the basis of Schvartzman's second model (1979) as well as Miller's model (1976), depending on the pattern of uptake of BrdU over three cell cycles. An interference in the previous events of exchanges in the following cell cycle (i.e., cancellation of SCEs) in BS chromosomes was observed in some regions, though not in high numbers.     

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.     

Sister chromatid differentiation and exchanges in adult mudminnows (Umbra limi) after in vivo exposure to 5-bromodeoxyuridine

An in vivo system for the detection of sister chromatid exchange (SCE) in the central mudminnow, Umbra limi, is presented. Sister chromatid differential (SCD) and SCE were demonstrated by fluorescent and Giemsa procedures 5 to 6 days after the fish were injected with 500 g/g of BrdU. The exchange rate was found to be 2.64 SCEs metaphase in the intestines and 2.42 SCEs/metaphase in the gills. SCE analysis in U. limi should be a useful tool for measuring the mutagenicity of water-borne chemicals.     

In vivo BrdU-33258 Hoechst analysis of DNA replication kinetics and sister chromatid exchange formation in mouse somatic and meiotic cells

BrdU (5-bromodeoxyuridine)-33258 Hoechst methods have been adapted for in vivo analyses of replication kinetics, sister chromatid differentiation and sister chromatid exchange (SCE) formation in mice. Sufficient in vivo BrdU substitution for cytological detection was effected with multiple intraperitoneal injections of the analogue. The combination of centromere staining asymmetry and sister chromatid differentiation at metaphase permits unambiguous determination of the number of replications in BrdU and dT (deoxythymidine) undergone by individual cells. Late-replicating regions in marrow and spermatogonial chromosomes are highlighted by bright fluorescence after sequential incorporation of BrdU followed by dT during a single DNA synthesis period. SCEs are analyzed in marrow and spermatogonial metaphases after successive complete cycles of BrdU and dT incorporation. Significant induction of SCE was observed with both mitomycin C and cyclophosphamide; the latter drug requires host-mediated activation to be effective. In meiotic metaphase cells harvested two weeks after BrdU incorporation, satellite DNA asymmetry, sister chromatid differentiation and SCE could be detected in a few chromosomes, most frequently the X and the Y.     

The distribution of mitomycin C-induced sister chromatid exchanges in the euchromatin and heterochromatin of the Indian Muntjac

The frequency of sister chromatid exchanges (SCEs) induced by mitomycin C (MMC) in Indian Muntjac chromosomes was determined by the fluorescence plus Giemsa (FPG) technique. Using scanning cytophotometry the relative DNA content of each chromosome was measured with and without acid or alkali pretreatments for C-banding. During acid and alkali treatments, euchromatin lost 20 to 30% of its DNA, while heterochromatin lost less than 5%; an intermediate DNA loss was observed for the short arm of the X chromosome. After growth of cells in the presence of MMC during the first cycle and in the presence of bromodeoxyuridine (BrdU) during the first and second cycles of DNA replication, SCEs in the euchromatin were proportional to DNA content. SCEs at the junctions between the neck of the X chromosome and the long and short arms occurred more frequently than expected. A threshold effect for the induction of SCEs was observed in regions resistant to DNA extraction by acid and alkali treatments (i.e., the neck and short arm of the X chromosome). At high concentrations of MMC, the frequency of SCE at each junction appears to plateau at 0.5.     

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     

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.     

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.     

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.     

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.     

Sister chromatid exchanges in human cells and Chinese hamster cells: Evidence that the rate of sister chromatid exchanges is a function of ploidy

The frequency of sister chromatid exchanges (SCE) in the chromosomes of the diploidy and polyploidy of Chinese hamster cells and human cells has been studied using BUdR-DAPI (bromodeoxyuridine, 4′-6-diamidino-2-phenylindol) fluorescence. The rate of SCEs per cell under constant control conditions is in proportion to the ploidy levels. In addition, the frequency of SCEs observed in a given human chromosome (nos. 1) is also directly proportional to the number of such chromosomes presented in the cells. The mean of SCEs in human chromosome numbers 1 is very similar (0.46–0.48) for diploid, triploid, and tetraploid cells. The results suggest that the rate of SCEs is a function of cellular ploidy levels.     

The effect of continuous gamma irradiation on formation of sister chromatid exchanges (SCEs) and chromosomal aberrations in meristematic cells ofVicia faba

Germinated seeds ofVicia faba were continuously irradiated at low dose rate of gamma rays (0.05 Gy h-¹) up to a total accumulated dose of 2 Gy. The FPG (fluorescence plus Giemsa) technique of differential chromatid staining was used to monitor the frequency of sister chromatid exchanges (SCEs) in irradiated root tip meristem cells. The results of the experiments have demonstrated that SCE frequency is raised by continuous gamma irradiation only in plant cells containing BrdU in the chromosomal DNA. No effect concerning SCE formation was recorded at continuous irradiation of meristematic cells of Vicia faba with native, i. e. BrdU-nonsubstituted, DNA. In contrast to SCEs, a significant increase was found in the yield of chromosomal aberrations in all variants of irradiation.