SCE variability in lymphocytes and fibroblasts

Summary To determine whether the sister chromatid exchange (SCE) distributions obtained in lymphocytes and fibroblasts from different individuals are comparable, a controlled study was set up. Peripheral blood and skin biopsies were taken on the same day from five individuals living for years under the same environmental conditions. All samples were treated in the same fashion, and the SCEs were scored in 50 metaphases of peripheral blood lymphocytes and of skin fibroblasts in an early and in a late passage. A repeat blood sample was taken from the same five indivuduals 1 year later. Based on the results obtained in this first part of the study, five randomly chosen healthy blood donors were sampled at different times and studied in the same fashion. Each chromosome was identified, and the SCE scores were tabulated per chromosome over 50 metaphases. The statistical analysis consisted of fitting log linear models to these scores and examining the best fit by determining the exceedance probabilities (observed significance level). For lymphocytes, the results indicated that the SCE distributions depended only on the chromosome examined, and not on BrdU-exposure time, individuals, or time of sampling. Treatment with ethyl methane sulfonate (EMS) increased the number of SCEs proportionally on all chromosomes. Analysis of the SCE scores on lymphocytes and fibroblasts of the five individuals and on their low and high passage fibroblast cultures revealed the necessity of including higher order interactions in order to fit a suitable model to the data. Therefore comparison of the SCE scores of lymphocytes with those of fibroblasts or comparison of scores on fibroblasts from different individuals could not be done. In practice, to compare samples or individuals, it suffices to score the SCE on a limited number of chromosomes (e.G., the A group) of 50 metaphases.     

Positional control of the distribution of spontaneous sister chromatid exchanges along mouse chromosomes]

The use of a new method having combined C-band staining and differential staining of sister chromatids allowed to determine a pattern of distribution of spontaneous sister chromatid exchanges (SCE) along cytologically marked chromosomes 1, 2 and 6 of house mouse. All chromosomes displayed the same pattern of SCE distribution: SCEs are most frequent in the middle part of the chromosome arm and rather rare near the centromere and the telomere. It has been suggested that this pattern of distribution is positional, rather chromatin-specific. The chromosome 1 carrying paracentric inversion with breakpoints in the middle part of the arm and just near the telomere has the same pattern of SCE distribution as normal chromosome 1. Double insertion of homogeneously staining regions in the middle part of the chromosome 1 produces increase in the SCE number per chromosome proportional to the physical length of the insertion. In contrast to meiotic recombination, interference between SCEs is not detected. No evidence for existence of the hot-spots of SCE on the junctions between C-positive and C-negative regions, as well as between G-bands and R-bands, has been produced.     

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.     

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.     

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.     

Sister chromatid exchange points in the heterochromatin and euchromatin regions of Chinese hedgehog chromosomes

Summary The Chinese hedgehog has a diploid chromosome number of 48 in which there are eleven pairs of telo- or subtelocentric autosomes, twelve pairs of meta- or submetacentric autosomes, a metacentric X chromosome and a telocentric Y chromosome. The heterochromatin is almost completely distributed in five large distal segments of chromosomes nos. 9 to 12 and no. 18. There is no positive C-band in the centromeres of the chromosomes except for the X chromosome which has a small, weakly stained C-band in the centromere. In Chinese hedgehog cells 52.1% of SCEs are found at the junction between the euchromatin and the heterochromatin, 39.5% in the heterochromatin and 8.4% in the auchromatin. The SCE number per unit C-band is double the SCE number per unit euchromatin. The SCE rate in the heterochromatin or euchromatin regions is not proportional to their chromosome length and can be quite different between different pairs of the chromosomes. Our results indicate that there is a non-uniform distribution of the SCEs in the Chinese hedgehog cells.     

Sister chromatid exchange distribution in various human tissues exposed to MMC and BrdU

The effect of two known mutagens on different human tissues was examined in an attempt to determine if tissue specific responses exist in SCE distribution on chromosome. The tissues included human lymphocytes, skin fibroblasts, ovarian and testicular cells. All cell types were exposed to varying concentrations of 5-bromodeoxyuridine (BrdU), and mitomycin C (MMC). The numbers of SCEs were recorded from each tissue. Results indicated that certain of the tissues tested appeared more sensitive to particular agents. Results also showed that in all the tissues tested the larger chromosomes in groups A to C had greater numbers of SCEs than did the smaller chromosomes in groups D to G. There were very few SCEs in F and G group chromosomes including Y.     

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.     

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     

Analysis of spontaneous sister-chromatid exchanges in vivo by three-way differentiation

By applying an adaptation of the method of three-way differentiation to murine bone marrow cells in vivo, the basal frequency of sister-chromatid exchange (SCE) per cell was evaluated. An SCE frequency directly proportional to the estimated relative incorporation of 5-bromodeoxyuridine (BrdU) to the chromosomes was observed for the 3 consecutive cell cycles, implying that the majority, if not all, of the SCEs in vivo were produced by the incorporated BrdU. This conclusion was supported by the finding that in the first cycle of division, a very high frequency of cells without SCE was observed. From these data, a spontaneous frequency of SCE as low as 0.15 SCE/cell/cell cycle was inferred.     

Sister chromatid exchanges in the human active and inactive X chromosomes

Four human female fibroblast strains with an i(Xq) or derivative X chromosome as a cytological marker for the inactive X chromosome were used to determine the frequency of sister chromatid exchanges (SCEs) in the active and inactive X chromosomes. No significant difference in SCE frequency between the active and inactive X chromosomes was observed. Therefore, the state of chromatin condensation and the late DNA replication in the facultative heterochromatin of the inactive X chromosome do not appear to influence the SCE frequency.     

Lack of Spontaneous Sister Chromatid Exchanges in Somatic Cells of DROSOPHILA MELANOGASTER

Neural ganglia of wild type third-instar larvae of Drosophila melanogaster were incubated for 13 hours at various concentrations of BUdR (1, 3, 9, 27 micrograms/ml). Metaphases were collected with colchicine, stained with Hoechst 33258, and scored under a fluorescence microscope. Metaphases in which the sister chromatids were clearly differentiated were scored for the presence of sister-chromatid exchanges (SCEs). At the lowest concentration of BUdR (1 microgram/ml), no SCEs were observed in either male or female neuroblasts. The SCEs were found at the higher concentrations of BUdR (3, 9, And 27 micrograms/ml) and with a greater frequency in females than in males. Therefore SCEs are not a spontaneous phenomenon in D. melanogaster, but are induced by BUdR incorporated in the DNA. A striking nonrandomness was found in the distribution of SCEs along the chromosomes. More than a third of the SCEs were clustered in the junctions between euchromatin and heterochromatin. The remaining SCEs were preferentially localized within the heterochromatic regions of the X chromosome and the autosomes and primarily on the entirely heterochromatic Y chromosome.--In order to find an alternative way of measuring the frequency of SCEs in the Drosophila neuroblasts, the occurrence of double dicentric rings was studied in two stocks carrying monocentric ring-X chromosomes. One ring chromosome, C(1)TR94--2, shows a rate of dicentric ring formation corresponding to the frequency of SCEs observed in the BUdR-labelled rod chromosomes. The other ring studied, R(1)2, exhibits a frequency of SCEs higher than that observed with both C(1) TR94--2 and rod chromosomes.     

Preferential occurrence of sister chromatid exchanges at heterochromatin-euchromatin junctions in the wallaby and hamster chromosomes

Chromosomes of two mammalian species, the white-throated wallaby and the rat-like hamster, possessed large amounts of constitutive heterochromatin which is detectable as C bands. By making use of this character, the frequency of sister chromatid exchanges (SCEs) was determined for the C band and the euchromatic regions of the chromosome. In both species, the distribution of SCEs in the euchromatin of chromosomes was found to be proportional to its metaphase length, while the number of SCEs localized in the C band regions was clearly fewer than expected on the basis of the relative length of those regions at metaphase. Many SCEs were, however, detected at the junctions between the euchromatin and the C band heterochromatin. All of these findings were consistent with previous observations on the Indian muntjac and the kangaroo rat chromosomes.     

Chromosome-damaging effects of heraclenin in human lymphocytes in vitro

Heraclenin, a furocoumarin with an epoxide group in its side chain, was analyzed to see if it induced structural chromosome aberrations and sister-chromatid exchanges (SCEs) in human lymphocytes in vitro. The results were compared directly with those of imperatorin, which differs from heraclenin only in lacking an epoxide group. An equally strong clastogenic effect was found for both heraclenin and imperatorin: the number of metaphases with breaks was increased in both cases by approximately a factor of 6. Heraclenin produced a considerable dose-dependent increase in the SCE rate, i.e., by about 60 induced SCEs/metaphase, whereas imperatorin induced only about 4 SCEs/metaphase. The results are discussed with respect to the occurrence of structural aberrations, which are primarily due to the basic furocoumarin structure itself, whereas the large increase in the SCE rate produced by heraclenin is most probably significantly influenced by its epoxide group.     

Sister-chromatid exchanges and chromosal breakage in patients treated with cytostatics.

The frequency of structural chromosomal rearrangements and sister-chromatid exchanges (SCEs) was investigated in short-term phytohemagglutininstimulated lymphocyte cultures by means of bromodeoxyuridine substitution and fluorescence plus Giemsa (FPG) staining technique. Both these parameters were significantly increased in patients treated with comparatively low doses of cyclophosphamide, busulphan and adriamycin. The increased SCE rate was proportional to the number of chromosome breaks, the ratio of SCE to breaks being about 100:1. The increase in the SCE number was maintained for several months after the termination of cytostatic therapy, when the conventional analysis of chromosome breaks yielded normal results. Normal SCE values were obtained in two patients treated with low doses of fluorouracil.     

Effects of caffeine on chromosome aberrations and sister-chromatid exchanges induced by mitomycin C in BrdU-labeled human chromosomes.

The BrdU-Hoechst staining technique has been used in analyzing the effect of caffeine (CAF) on chromosome aberrations and sister-chromatid exchanges (SCEs) induced by mitomycin C (MC). CAF increased the frequency of SCE in MC-treated chromosomes in all specimens. The combination of MC and CAF caused a remarkable increase in all types of chromosome aberrations, but the most startling effect was the appearance of many cells with multiple aberrations (shattered chromosomes). The BrdU-Hoechst technique showed that the shattered chromosomes did not appear in cells that had replicated only once, but did occur in cells which replicated twice in the presence of MC and CAF. The large majority of chromatid breaks observed did not involve areas common to SCE; and the SCE frequency significantly increased in spite of the existence of multiple breaks. This indicates that very few of the breaks are incomplete exchanges and that the mechanism for formation of SCE might be different from that of chromosome breaks. In another experiment, monofunctional-MC (M-MC) had a small effect on SCE rates, though it induced shattered chromosomes with CAF post-treatment. Possible differences in the mechanisms leading to SCE and chromosome breaks are discussed.     

Anthramycin-induced sister-chromatid exchange and caffeine potentiation in the chromosomes of Indian muntjac

Cell-cycle kinetics, sister-chromatid exchange (SCE) and chromosome aberrations have been studied from the skin fibroblasts of the Indian muntjac after treatment with 100 micrograms/ml of caffeine and 0.05 microgram/ml of anthramycin. The cultures were incubated for a period which was sufficient for the completion of two consecutive cell cycles and both the drugs appeared to produce a slight inhibitory effect. When anthramycin-treated cells were however post-treated with caffeine, the cells did not proceed beyond one cycle and exhibited a mitotic block. The SCE frequency in the control and the experiments with caffeine and anthramycin was 8.63, 18.32 and 34.88 per cell respectively. The SCEs were randomly distributed amongst all chromosomes unlike a non-random distribution within the X chromosomes. Caffeine and anthramycin produced only 0.5% and 3.1 cells with chromosome aberrations respectively. Potentiation of chromosome aberrations was observed when the anthramycin-treated cells were post-treated with caffeine. Caffeine potentiation presumably results from an inhibition of the cells to cycle and a failure to repair the effect of the mutagen on DNA.     

Frequent occurrence of UVB-induced sister chromatid exchanges in telomere regions and its implication to telomere maintenance

Sister chromatid exchanges (SCEs) are symmetrical exchanges between newly replicated chromatids and their sisters. While homologous recombination may be one of the principal mechanisms responsible for SCEs, the full details of their molecular basis and biological significance remain to be elucidated. Following exposure to ultraviolet light B (UVB), mitomycin C (MMC) and cisplatin, we analyzed the location of SCEs on metaphase chromosomes in Chinese hamster CHO cells. The frequency of SCEs increased over the spontaneous level in proportion to the agent's dose. UVB-induced SCEs occurred frequently in telomere regions, as cisplatin-induced SCEs did, differing from MMC-induced ones. The remarkable difference of intrachromosomal distribution among the three mutagens may be attributed to the specificity of induced DNA lesions and structures of different chromosome regions. Telomeric DNA at the end of chromosomes is composed of multiple copies of a repeated motif, 5'-TTAGGG-3' in mammalian cells. Telomeric repeats may be potential targets for UVB and cisplatin, which mainly form pyrimidine dimers and intrastrand d(GpG) cross-links, respectively, resulting in SCE formation. UVB irradiation shortened telomeres and augmented the telomerase activity. The possible implications of the frequent occurrence of SCEs in telomere regions are discussed in connection with the maintenance of telomere integrity.     

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.