Sister Chromatid Differentiation Research Articles

Genotoxic Effect of Dye Effluents in Chromosomes of Indigenous Goats (Capra hircus)

The genotoxic effect of dye effluents was assessed in indigenous goats inhabiting textile industry areas encompassing dyeing and bleaching units by means of mitotic drive, mitotic index, chromosome aberrations and the mean SCEs frequency. Peripheral blood leucocyte cultures were set up with culture medium and pokeweed mitogen. The cultures were supplemented without bromodeoxyuridine (BrdU) for chromosome aberrations and with BrdU for the mean SCEs frequency for 2 cell cycles. Air-dried slides were stained with Giemsa for chromosome aberrations and the fluorescent plus Giemsa (FPG) technique was adopted for the display of sister chromatid differentiation and the estimation of the mean SCEs frequency. No gross structural and numerical chromosome abnormalities were noticed in all animals screened based on G-banding and karyotyping. The mitotic drive for the control and exposed populations was 37.58±1.29 and 26.13±0.78%, respectively. The mitotic drive in exposed population was significantly (p<0.01) lower when compared to control animals. The mitotic index for the control and exposed populations was 12.49±0.42 and 11.49±0.43%, respectively. Though there was a decrease in mitotic index in the exposed population, it was not statistically significant. The frequency of SCEs did not follow the Poisson distribution in both the control and exposed populations of goats. The pooled mean±S.E (range) of SCEs frequency for the control and exposed populations of goats were 4.83±0.16 (1–11) and 12.98±0.16 (8–19), respectively. There was significant (p<0.01) increase in the mean SCEs frequency of goats (exposed) reared in industrial areas. The number of SCEs in chromosome 1 was significantly (p<0.05) higher than that expected from relative chromosomal length.

Further characterization of the genotoxicity of formaldehyde in vitro by the sister chromatid exchange test and co-cultivation experiments

The induction of sister chromatid exchanges (SCE) was used to further characterize the genotoxic action of formaldehyde (FA) on cultured mammalian cells. FA induced SCE in V79 Chinese hamster cells and A549 human lung cells in a concentration-related manner. Addition of 5-bromodeoxyuridine (BrdUrd) for the differentiation of sister chromatids to visualize SCE 4 h after the FA treatment led to a clearly reduced induction of SCE in agreement with the repair kinetics of FA-induced DNA-protein cross-links. When A549 cells were treated with FA for 1 h and then co-cultivated with V79 cells in the presence of BrdUrd, a clear induction of SCE was measured in V79 cells. When the same experiment was performed including washing and change of medium after the FA treatment, no induction of SCE was measured in V79 cells. These results indicate that reactive FA remains in the cell culture medium for a longer time period despite the high reactivity of FA with macromolecules. However, FA that has entered a cell is not released and does not damage other cells. Possible implications for the mutagenicity of FA in vivo will be discussed.

Analysis of SCEs in Vicia faba chromosomes by a simple fluorescent plus Giemsa technique

Cortés, F. and Andersson, H.C. 1987. Analysis of SCEs in Viciafaba chromosomes by a simple fluorescent plus Giemsa technique. —Hereditas 207: 7–13. Lund, Sweden. ISSN 0018–0661. Received October 3, 1986 A modified fluorescent plus Giemsa (FPG) technique for differential staining of sister chromatids and for studying sister chromatid exchanges (SCE) in root tip cells of Viciafaba is described. This new technique is quick and simple and has the advantage of a high reproducibility. The best quality of the sister chromatid differentiation (SCD) is obtained when seedlings are grown for two rounds of chromosome replication in the presence of 5-bromodeoxyuridine (BrdUrd). The technique stains the bifilarly substituted chromatid light and the unifilarly substituted one dark. However. SCD is also obtained after one round of replication in BrdUrd followed by another in the presence of thymidine. There was no difference in the baseline level of SCE in the two types of experimental protocol. The influence of various concentrations of BrdUrd on the yield of SCEs was investigated, as well as the frequency of SCEs in root tip cells grown in the absence and presence of 5-fluorodeoxyuridine. The results of these experiments are discussed.

Mechanism of in vivo sister-chromatid exchange induction by 5-azacytidine

The aim of the present study was to explore the in vivo mechanism of sister-chromatid exchange (SCE) induction by 5-azacytidine (5-azaC) in murine bone marrow cells. Experiments were performed to examine SCE induction in response to different doses of 5-azaC as well as several exposures. Additionally, we examined the persistence of SCE induction and the effect of bromodeoxiuridine (BrdU) incorporation. Sister-chromatid differentiation was obtained by injecting mice intraperitoneally with BrdU absorbed to activated charcoal. Before BrdU injection, different doses of 5-azaC were administered intraperitoneally either singly or in multiples. Colchicine in an aqueous solution was administered subcutaneously 22 h after BrdU injection. Two hours later, animals were sacrificed by cervical dislocation and both femurs were dissected. Bone marrow cells were processed to obtain chromosome preparations, which were stained by the fluorescence plus Giemsa method. Results indicate that 5-azaC caused SCE, albeit to a limited extent. In order to discern whether the limitation was due to cytotoxicity or to partial 5-azaC incorporation, we administered multiple sub-toxic doses of 5-azaC. This treatment increased 5-azaC incorporation and reduced cytotoxicity, but did not raise SCE frequency, indicating that the limitation was not due to either of the two factors mentioned above. SCE frequency induced by 5-azaC persisted for at least eight cell divisions, confirming that this agent had caused inhibition of DNA methyltransferase and subsequently the reduction of DNA re-methylation, which in turn induced the expression of a number of SCE-prone sites. Finally, SCE induction in response to 5-azaC was completely dependent on BrdU incorporation. The data allow us to conclude that 5-azaC causes SCE to a limited extent; limited SCE induction was not due to the direct effect of incorporation or cytotoxicity of 5-azaC, but rather the generation of a number of SCE-prone sites, the expression of which persists for at least eight cell divisions and is dependent on BrdU incorporation.

Cell cycle and centromere FISH studies in premature centromere division

BackgroundMitotic configurations consistent in split centromeres and splayed chromatids in all or most of the chromosomes or premature centromere division (PCD) have been described in three categories. (1) Low frequency of PCD observed in colchicines-treated lymphocyte cultures from normal individuals. (2) High frequency of PCD with mosaic variegated aneuploidy. (3) High frequency of PCD as a sole chromosome abnormality observed in individuals with no recognizable clinical pattern. We report four members of a family with the third category of PCD.MethodsCell cycle duration assessed by average generation time using differential sister chromatid stain analysis and FISH studies of DNA centromere sequences in PCD individuals, are included and compared with previously reported PCD individuals from 9 families.ResultsWe observed PCD in colchicine-treated cultures from the propositus, his father, and two paternal aunts but not in his mother and four other paternal and maternal family members, as well as in untreated cultures from the propositus and his father. We observed cytological evidence of active centromeres by Cd stain. Significative cell cycle time reduction in anaphases of PCD individuals (average generation time of 21.8 h;SD 0.4) with respect to individuals without PCD (average generation time of 31.8 h;SD 3.9) was observed (P < 0.005, Student t-test for independent samples). Increased cell proliferation kinetics was observed in anaphasic cells of individuals with PCD, by differential sister chromatid stain analysis. FISH studies revealed the presence of alpha satellite DNA from chromosomes 1, 13, 21/18, X, all centromeres, and CENP-B box sequences in metaphasic and anaphasic cells from PCD individuals.ConclusionThis report examines evidences of a functional relationship between PCD and cell cycle impairment. It seems that essential centromere integrity is present in these cases.

Sister chromatid differentiation with biotin-dUTP.

The method of choice to differentiate sister chromatids is to incorporate BrdU in replicating DNA. The disadvantage of BrdU is that its spontaneous or induced radicalization may itself lead to sister chromatid exchanges. Biotin-labelled dUTP is a widely used thymidine analogon for labelling isolated DNA. Its chemical structure suggests that, in contrast to BrdU, it does not give rise to radical formation. We electroporated proliferating Chinese hamster ovary (CHO) cells in the presence of biotin-dUTP which was subsequently detected in metaphase cells with TRITC-conjugated avidin. Microscopic analysis of second mitoses after labelling revealed a clear differential staining of sister chromatids. Thus substitution of thymidine with biotin-dUTP is another method to analyse SCE.

The influence of demethylating agents on preimplantation development of mice.

The effects of two demethylating drugs with a different mechanism of action (5-azacytidine (Aza) and L-ethionine (Eth)) on mouse preimplantation development were investigated. Preimplantation embryos were cultured for 24 h in the presence of the drug and for an additional 24 or 48 h (depending on the cleavage stage) in medium supplemented with bromodeoxyuridine to reveal sister chromatid exchanges (SCEs) and the number of cell cycles performed before harvesting. Striking differences between the two drugs were observed in their influence on proliferation of blastomeres, primary differentiation and sister chromatid differentiation (SCD), and in the pattern of DNA methylation and the frequency of SCEs per cell. At a final concentration of 1 microM Aza had no effects, whereas higher concentrations stopped development of all stages except the zygote. In contrast Eth treatments (5 mM) resulted in a severe reduction of the mean cell number per embryo in comparison with controls. Moreover both the absence of blastocyst formation and no effects on mitotic activity were detected. The most prominent effect of Eth was detected at the zygote and 4-cell stages. An unexpected decrease in SCE frequency in Eth-treated morulae and 4-cell embryos has been observed. Data are explained taking into account the different mechanisms of action of the agents.

Differential sister chromatid exchange response in phytohemagglutinin and pokeweed stimulated lymphocytes of goat (Capra hircus L)

Une reponse differentielle pour le nombre d'echanges entre chromatides soeurs a ete observee entre les lymphocytes de chevre (Capra Hircus L) stimules par la phytohemagglutinine (PHA) ou le phytolaque (PKW), apres exposition a des concentrations finales de 0,1, 0,25, 0,5, 1, 2,5 et 5 μg/mL de BUdR. A 0,1 μg/mL de BUdR, les deux mitogenes ont donne une reponse SCE/cellules tres similaire: les valeurs moyennes ont ete de 3,17 ± 1,93 pour PHA et de 3, 28 ± 1,76 pour PKW, et les distributions de frequences se sont tres bien ajustees a une loi de Poisson pour les deux mitogenes. A partir de 0,25μg/mL, les reponses a PKW ont toujours ete superieures a celles correspondant a PHA. A partir de 5 μg/mL de BUdR, la reponse SCE/ cellule a ete de 8,68 ± 3, 24 pour PKW et 6, 96 ± 3, 45 pour PHA et la difference a ete significative. La distribution de frequence pour l'ensemble des mitogenes s'est tres bien ajustee a un melange de deux lois de Poisson, correspondant aux deux populations de cellules concernees par chaque mitogene.

Differential sister chromatid exchange response in phytohemagglutinin and pokeweed stimulated lymphocytes of goat (Capra hircus L)

Differential sister chromatid exchange response in phytohemagglutinin and pokeweed stimulated lymphocytes of goat (Capra hircus L)

Investigating the nature of chromatid breaks produced by restriction endonucleases

It is a basic assumption of the breakage-and-reunion theory that the majority of open chromatid breaks seen at metaphase are the residue of unrejoined primary breaks that have neither restituted nor rejoined illegitimately to form exchange aberrations. If Chinese hamster chromosomes with BrdU sister-chromatid differentiation are irradiated, and chromatid aberrations scored from G2 cells, some 15-20 of open breaks show a colour-jump at the point of discontinuity, indicating a two-lesion intrachange origin. Since we see complete forms of several intrachanges whose incomplete forms will also look like breaks, but devoid of a colour-jump, it appears that a substantial proportion of observed breaks are intrachange derived. Experiments to date show that the colour-jump proportion is constant, irrespective of radiation dose, radiation quality, BrdU concentration and hamster cell origin. It is the same for the very low 'spontaneous' breaks found in control samples. Restriction endonucleases RE can be introduced into cells by various poration methods, and are highly efficient at producing all types of aberrations. This is taken as strong evidence that DNA dsb are significant lesions triggering aberrations. One might anticipate, therefore, that observed breaks will be predominantly unrejoined dsb, and the proportion of colour-jump breaks correspondingly low. We tested this supposition using three RE; Alu I, a blunt-end cutter, Sau3A I, a cohesive-end cutter, both with a short life-time in vivo, and Mbo I, an isoschizomer of Sau3A I, which has a long cutting life-time in vivo. Although there were differences in absolute yields of breaks, and of relative frequencies of aberration types recovered, the proportion of colour-jump breaks was as high as that in a parallel X-ray experiment, and fell well within the range encountered in all our previous experiments. It is difficult to reconcile this universal constancy of colour-jump breaks with the expectations of breakage-and-reunion theory, where the occurrence of two-break events must be a treatment variable. Rather, our results suggest that most open breaks are secondary, resulting from a regular intrachange processing mechanism.

Do chiasmata disappear? An examination of whether closely spaced chiasmata are liable to reduction or loss

The questions of whether closely spaced crossovers could be misidentified as single chiasmata or could cancel out each other and whether exchange could occur without chiasma formation were examined in Locusta migratoria. Monochiasmate bivalents that showed differential sister chromatid staining following bromodeoxyuridine incorporation were screened for their patterns of label distribution. Half of the chiasmata were associated with an exchange between dark and lightly stained chromatids, as expected if recombination involved any two non-sister chromatids chosen at random. Two variant types of label distribution were also seen: approximately 10% of all monochiasmate bivalents had an anomalous distribution of dark and light chromatids around the chiasma, and in three of the 1365 bivalents screened a second type of anomalous pattern was observed for the first time, in which all four chromatids had a label exchange at the chiasma. The observed incidence of the latter was considerably less than expected if they originated through a four-strand double crossover with closely positioned exchanges. Analysis of label distribution patterns in monochiasmate bivalents did not produce evidence of the other configurations expected if two closely spaced exchanges could be misidentified as single chiasmata or could cancel one another out so as not to form a chiasma. We conclude that analysis of chiasma frequencies and distributions offers an accurate means of assessing recombination in organisms with favourable cytology.

Chemical induction of interleukin-8, a proinflammatory chemokine, in human epidermal keratinocyte cultures and its relation to cytogenetic toxicity

Tumor promoters, proinflammatory cytokines, endotoxins, and protein synthesis inhibitors can modulate cell cycle kinetics of various cell types, stimulate production of reactive oxygen species, and induce keratinocytes to produce interleukin-8 (IL-8), a potent chemotactant for polymorphonuclear neutrophils and T lymphocytes. The aim of this study was to determine whether perturbations of cytogenetic responses correlated with the induction of IL-8 expression. Cultures of primary human keratinocytes were grown in serum-free medium with 5 mumol/L bromodeoxyuridine to label DNA and exposed either to phorbol-13-myristate-12-acetate (PMA) (0.0001-100 ng/ml), cycloheximide (CHX) (0.01-50 micrograms), lipopolysaccharide (0.1-100 micrograms/ml), tumor necrosis factor-alpha (TNF alpha) (3.13-50 ng/ml), or interleukin-1 alpha (IL-1 alpha) (1-182 pg/ml). Metaphase chromosome preparations were stained by a fluorescence-plus-Giemsa technique to differentiate sister chromatids. For IL-8 production, keratinocytes were grown to 70% confluency and then exposed to chemicals for 24 h. Immunoreactive IL-8 was quantitated from the supernatants by ELISA. With the exception of benzo(a)pyrene used as a positive control, none of the agents induced sister chromatid exchanges. However, PMA and TNF alpha induced IL-8 production that coincided with significant cell cycle inhibition. IL-1 alpha had no effect on cytogenetic endpoints, yet stimulated a 6.3-fold increase in IL-8. CHX inhibited cell cycle progression and mitotic activity at concentrations that were 200 times lower than required for IL-8 induction; however, puromycin (0.31-10 micrograms/ml), another protein synthesis inhibitor, did not induce IL-8. At all concentrations tested, TNF alpha reduced the mitotic index by approximately 45%, slowed cell cycle progression by approximately 3.5 h, and induced a flat, albeit large, IL-8 response at concentrations > or = 12.5 ng/ml. These agent-specific response patterns suggest that induction of IL-8 production is not always the inevitable result of cell cycle perturbations or genetic damage.

Detection of sister chromatid exchange sites by sequential staining and imaging methods.

A simplified fluorescence plus giemsa method for demonstrating sister chromatid differentiation has been developed in conjunction with a C-banding method for the same barley metaphase chromosomal spread. By combining this method with imaging methods, the sites of sister chromatid exchanges could be exactly determined.

Combined FISH painting and harlequin staining for cell cycle-controlled chromosome analysis in human lymphocytes.

We present a simple method that allows scoring of FISH-painted chromosomes exclusively in the first division human lymphocytes. It consists of a combination of FISH with chromosome-specific libraries and a differential sister chromatid staining after BrdU treatment of lymphocyte cultures. The method allows a precise quantification of induced chromosome damage for human biodosimetry.

Sister chromatid differentiation in 5-bromo-2?-deoxyuridine-substituted chromosomes: A study with DNA-specific ligands and monoclonal antibody to histone H2B

Human and Chinese hamster chromosomes were obtained from cells grown in the presence of 5-bromodeoxyuridine (BrdU) for (a) one replicative round, (b) two replicative rounds, (c) one replicative round followed by another round in thymidine and (d) the last period of synthetic phase. Untreated chromosomes and chromosomes treated with UV radiation after previous staining with 33258 Hoechst as photosensitizer were studied in order to investigate the mechanism(s) responsible for BrdU-induced sister chromatid differentiation (SCD). Metaphases were prepared by (1) standard methanol-acetic acid fixative for subsequent investigation with Giemsa or DNA-specific dyes such as ethidium bromide, acridine orange and monoclonal antibodies to double- or single-stranded DNA; (2) the procedure for observation under phase-contrast or electron microscopy; and (3) the cytospin method for subsequent immunoreaction with a monoclonal antibody to histone H2B. Our data exclude the possibility that the presence/absence of BrdU in the template strand might affect chromatin organization and thus resistance, while confirming that UV-induced DNA alteration is not sufficient, by itself, to explain SCD mechanism(s). That molecules other than DNA play a role in explaining SCD production is indicated by the fact that BrdU incorporation induces alterations in DNA-histone H2B interactions which, in turn, seem to produce structural variations in chromatin, possibly at the level of condensation, as monitored by phase-contrast and electron microscopy.

Proliferation kinetics of mussel (Mytilus galloprovincialis) gill cells

A technique of sister chromatid differentiation (SCD) using bromodeoxyuridine (BrdU) incorporation and a modification of the fluorescence plus Giemsa (FPG) method was employed to determine cell-proliferation kinetics in gill tissue of the mussel Mytilus galloprovincialis. Dose-dependent proliferation inhibition was examined. In vivo administration of BrdU for 12, 24, 36, 48, 60, 72, 84, and 96 h was studied. Our data show that the highest yield of second-generation metaphase plates is obtained after 60 h BrdU treatment; the duration of a cell cycle is 24 to 30 h. On the basis of these data, a BrdU incorporation period of 48 to 60 h would seem to be most appropriate for the sister chromatid exchange (SCE) tests carried out in gill cells of M. galloprovincialis, whereas the 12 to 24 h exposure would give the best results for replication band analysis.

Evaluation of sister-chromatid exchanges in mouse spermatogonia: a comparison between the classical fluorescence plus Giemsa staining and an immunocytochemical approach

Sister-chromatid exchanges (SCE) were analyzed in mouse spermatogonia using two different protocols for bromodeoxyuridine (BrdU) exposure and detection. With the classical approach, based on subcutaneous implantation of agar-coated BrdU tablets and fluorescence plus Giemsa (FPG) staining a satisfactory differentiation of spermatogonial metaphases was obtained with 50 or 25 mg BrdU per mouse (two or one tablets respectively). Alternatively, the immunodetection of BrdU was carried out after exposure to a very low BrdU concentration (three injections i.p., 3 mg/kg b.w. each, at 5-h intervals), and after exposure to one BrdU tablet; SCE frequencies evaluated in this way were lower than those found after classical FPG staining, even when the mice were exposed to the same BrdU concentration (one tablet, 25 mg BrdU). We concluded that the two methodologies may have different sensitivities with respect to SCE detection. In addition, when the effect of a treatment with mitomycin C was tested (1 mg/kg b.w, at time intervals ranging from 24 h to 5 days), no sister-chromatid differentiation was obtained with the multiple injection protocol, or with one BrdU tablet. By contrast, with two BrdU tablets and FPG, well differentiated metaphases were found at any time interval tested after MMC treatment, and the peak frequency of SCE (3.4 times the baseline) was observed at 55 h after treatment, as expected on the basis of cell cycle duration in spermatogonia. In summary, even though the same of medium-low concentrations of BrdU was successful in untreated animals, these protocols appeared inadequate to detect SCE induction by MMC. It is possible that, in the presence of cell cycle delay induced by the treatment, interferences with the rate of BrdU uptake produce an unsatisfactory differentiation of sister chromatids.

Sister chromatid differentiation after in situ detection of ultraviolet-induced DNA breaks under electron microscopy

Chinese hamster DON cells with 5-bromodeoxyuridine (BrdU)-substituted chromosomes were ultraviolet (UV)-exposed and processed for in situ detection of induced DNA breaks under electron microscopy. For this purpose, UV-induced breaks were amplified by an exonuclease III digestion to obtain single stranded DNA motifs which could hybridize with oligonucleotides of random sequences. These reannealed motifs could be used as primers which were extended by the Klenow polymerase, incorporating biotinylated-dUTP that was detected by a gold-tagged streptavidin. After processing, the chromatid whose DNA was BrdU-substituted in one strand showed a higher electron density than the chromatid substituted in both strands. In contrast, the unifilarly substituted chromatid showed about twice the labelling of DNA breaks as the bifilarly substituted one. This result could be the consequence of a greater loss of chromatin tracts in the bifilarly substituted chromatid, as implied by an X-ray microanalysis which showed that the amount of phosphorous lost by the bifilarly substituted chromatid was higher than that of the unifilarly substituted chromatid.

Spontaneous sister chromatid differentiation (SCD) and sister chromatid exchange (SCE) in mouse blastocyst chromosomes

The phenomenon of spontaneous differentiation (without bromodeoxyuridine in the culture medium) of sister chromatids of mouse chromosomes at the blastocyst stage of embryogenesis is described. The frequency of sister chromatid exchanges in such differentiated chromosomes was calculated.

A case of caffeine-mediated cancellation of mitotic delay without enhanced breakage in V79 cells

Chinese hamster cells (V79 379A) were grown for 17 h in the presence of 10 μg/ml bromodeoxyuridine (BrdU) to obtain cells with potential sister-chromatid differentiation. At this time batches were irradiated (1.5 Gy, 250 kVp X-rays) and the medium of all flasks replaced with one containing 10 μg/ml thymidine with or without 400 μg/ml caffeine. Metaphases from irradiated and unirradiated batches were sampled every hour for 7 h and FPG stained. All categories of chromatid-type aberrations were scored in G 2 and S phase cells. As expected, mitotic delay in the presence of caffeine, (measured by a shift in the portion of the fraction of differentially stained metaphases (FDM) curve and a reduced fall in the mitotic index) was largely cancelled, but there was a negligible increase in chromatid-aberration frequency (all categories), only achieving significance if the consistency of the whole 7-h sampling period was considered. Caffeine had no effect on the frequencies of light/dark chromatid involvement, nor in the completeness of chromatid interchanges. We conclude that the enhanced breakage frequency often observed with post-irradiation caffeine treatment is not necessarily causally related to the cancellation of delay.