SCE Analysis Research Articles

Sister Chromatid Exchange and Genomic Instability in Soft Tissue Sarcomas: Potential Implications for Response to DNA-Damaging Treatments.

Sarcomas are rare heterogeneous malignancies of mesenchymal origin characterised by complex karyotypes but no specific abnormalities. Recurrence is common, and metastatic disease carries poor survival despite standard DNA-damaging radiotherapy or chemotherapy. DNA double-strand breaks (DSBs) are either repaired by mechanisms such as homologous recombination (HR) or result in cell death by apoptosis. Endogenous γH2AX formation and SCE formation are early and late events, respectively, and their levels are considered surrogate measures of genomic instability. Combined γH2AX and SCE analysis was used to evaluate endogenous DNA DSB levels (and their subsequent repair) in 9 primary sarcoma cell lines and compared with well-established commercial lines. All the sarcoma cell lines had elevated γH2AX and SCE levels, but there was no correlation between the DNA DSB frequency and subsequent SCE. Typically, radioresistant osteosarcoma cells had relatively low γH2AX frequency but high SCE counts suggestive of efficient DNA repair. Conversely, liposarcoma cells derived from a radiosensitive tumour had high H2AX but relatively lower SCE levels that may imply inefficient DNA DSB repair. To our knowledge, this is the first report that correlates H2AX and SCE levels in primary sarcoma cell lines and may provide insight into potential response to DNA-damaging treatments.

The Assessment of Cytotoxicity and Genotoxicity of Tetracycline Antibiotic in Human Blood Lymphocytes Using CBMN and SCE Analysis, in Vitro

Tetracycline antibiotic, a widely used antimicrobial drug was tested for nuclear DNA damage in cultured peripheral blood lymphocytes in terms of chromosome alterations. The extent of cytogenetic da...

The Assessment of Cytotoxicity and Genotoxicity of Tetracycline Antibiotic in Human Blood Lymphocytes Using CBMN and SCE Analysis, in Vitro

The Assessment of Cytotoxicity and Genotoxicity of Tetracycline Antibiotic in Human Blood Lymphocytes Using CBMN and SCE Analysis, in Vitro

Lack of genotoxicity in medical oncology nurses handling antineoplastic drugs: Effect of work environment and protective equipment

In this study we aimed to investigate the genotoxic effects of antineoplastic agents in occupationally exposed oncology nurses. Genotoxic effects mean the disruptive effects in the integrity of DNA and they are associated with cancer development. Biomonitoring of health care workers handling antineoplastic agents is helpful for the evaluation of exposure to cytostatics. The study included an exposed and two control groups. The exposed group (n=9) was comprised of oncology nurses. The first (n=9) and second (n=10) control groups were comprised of subjects who did not come into contact with antineoplastic drugs working respectively in the same department with oncology nurses and in different departments. Genotoxicity evaluation was performed using SCE analysis. After applying culture, harvest and chromosome staining procedures, a total of 25 metaphases were analyzed per person. Kruskal Wallis test was used to perform statistical analysis. A statistically significant difference of sister chromatid exchange frequencies was not observed between the exposed and control groups. Lack of genotoxicity in medical oncology nurses might be due to good working conditions with high standards of technical equipment and improved personal protection.

Genotoxicity of the herbicide 2,4-dichlorophenoxyacetic and a commercial formulation, 2,4-dichlorophenoxyacetic acid dimethylamine salt. I. Evaluation of DNA damage and cytogenetic endpoints in Chinese Hamster ovary (CHO) cells

Genotoxicity of the 2,4-dichlorophenoxyacetic acid (2,4-D) and a commercially-used derivative, 2,4-D dimethylamine salt (2,4-D DMA), was evaluated in CHO cells using SCE and single cell gel electrophoresis (SCGE) assays. Log-phase cells were treated with 2.0–10.0 μg/ml of herbicides and harvested 24 and 36 h later for SCE analysis. Both agents induced significant dose-dependent increases in SCE, regardless of the harvesting time (2,4-D: r = 0.98 and r = 0.88, P < 0.01, for 24 and 36 h harvesting times; 2,4-D DMA: r = 0.97 and r = 0.88, P < 0.01, for 24 and 36 h harvesting times). Neither test compound altered cell-cycle progression or proliferative replication index ( P > 0.05), but the higher doses of both compounds reduced the mitotic index of cultures harvested at 24 and 36 h ( P < 0.05). A 90-min treatment with 2.0–10.0 μg/ml 2,4-D and 2,4-D DMA produced dose-dependent increases in the frequency of DNA-strand breaks detected in the SCGE assay, both in cultures harvested immediately after treatment and in cultures harvested 36 h later. The doses of 2,4-D and 2,4-D DMA were equally genotoxic in all of the assays. The results indicate that 2,4-D induces SCE and DNA damage in mammalian cells, and should be considered as potentially hazardous to humans.

Sister-chromatid exchange analysis on long-term benzathine penicillin for secondary prophylaxis of rheumatic fever.

A single intra-muscular injection of 1.2 millions units of benzathine penicillin every 4 weeks is the most widely used method for the antibiotic prophylaxis of rheumatic fever. The aim of this study is to evaluate the effect of long-term benzathine penicillin on DNA in patients with rheumatic fever. Thirty children with confirmed rheumatic fever who were on the benzathine penicillin prophylaxis were enrolled in the study, and 30 similar normal children served as a control group. To detect any DNA damage, SCE analysis were performed in circulating lymphocytes of the subjects. A statistically significant increased frequency of SCE was observed in children on the benzathine penicillin prophylaxis (no = 30, mean SCEs/cell +/- SD 7.54 +/- 1.81) as compared to a control group (no = 30, mean SCEs/cell +/- SD 5.82 +/- 1.40). It has been suggested that the difference in the SCE frequencies was induced by the administration of the benzathine penicillin for a long time, and further investigations are needed to confirm this toxic effect.

X-irradiation of G1 CHO cells induces SCE which are both true and false in BrdU-substituted cells but only false in biotin-dUTP-substituted cells.

The SCE-test is widely used in genetic toxicology and therefore knowledge of the contribution of BrdU to the formation of spontaneous and induced SCE is of great importance. The present study was undertaken to analyse the role of BrdU in X-ray-induced SCE. If SCE resulted from inversions, rings and double minutes (RDM) would be the asymmetrical counterparts of SCE and should therefore have the same frequencies. Dose-effect relationships of SCE and RDM show that the frequencies of SCE are much higher than those of RDM. We conclude that only a few SCE may represent inversions. In a second set of experiments, endoreduplications were induced in cells irradiated either before or after labelling with BrdU. Analysis of SCE in endoreduplicated chromosomes allows the discrimination of the cell cycle in which they originated. The results show that SCE are only induced in the first cell cycle following irradiation of BrdU-substituted cells, indicating that labelling with BrdU is a necessary prerequisite for the formation of SCE. In order to test this directly, radiation-induced SCE frequencies were studied in cells prelabelled with BrdU or biotin-dUTP in a third set of experiments. The structure of biotin-dUTP suggests that, in contrast to BrdU, it does not give rise to radicals during irradiation. Significantly lower frequencies of SCE were observed in biotin-dUTP-substituted cells than in BrdU-labelled cells. Calculations show that nearly all SCE induced in biotin-dUTP-labelled chromosomes can be explained by chromosomal aberrations (false SCE). In contrast to this, most SCE induced by X-rays in BrdU-labelled cells are not due to chromosomal aberrations, but result from S-dependent lesions (true SCE). This clearly points towards radiation damage in BrdU-moieties as the source of DNA lesions leading to SCE.

Comparative studies by comet test and SCE analysis in human lymphocytes from 200 healthy subjects

The comet test (single cell gel electrophoresis, SCGE) appears to be a promising tool to estimate DNA damage at the single cell level and it provides information on the presence of damage among individual cells. Previously, we analyzed the degree of DNA damage in peripheral human lymphocytes from 100 healthy subjects living in Pisa (Italy) taking into account age, gender and smoking habit, and we also reported some results aiming at the assessment of the comet test (Betti et al., 1994). In addition, SCE analysis was carried out in order to compare the two endpoints. Because of the interesting results obtained, the present study was extended to 200 individuals, and data analyzed included information concerning number of cigarettes smoked a day, tar/cigarette and job. Data obtained confirmed that the SCGE is more sensitive than SCE in revealing smoking habit effects but comet induction did not seem to be related to the amount of cigarette tar inhaled. Moreover, sampling time was found to play a greater role in the comet assay as compared to SCE. Job position did not significantly influence SCE mean/subject or comet length mean/subject.

Microgel electrophoresis assay (comet test) and SCE analysis in human lymphocytes from 100 normal subjects

Microscopic examination of individual human lymphocytes embedded in agarose, subjected to electrophoresis and stained with a fluorescent DNA-binding dye, provides a novel way of measuring DNA damage as extent of migration of DNA fragments, mainly single-strand breaks. With this relatively simple method, DNA damage arising as a consequence of smoking, age and other factors was examined in peripheral human lymphocytes from 100 healthy individuals living in Pisa (Italy). The extent of DNA migration was found to be significantly increased by smoking. It is noteworthy that the effect of smoking was more significant in men than in women and that DNA migration was similar in the young and in the older people. SCE analysis did not reveal any significant effect of smoking, sex or age in the same population, suggesting a higher responsiveness of the comet test to DNA-damaging agents.

Analysis of karyotype, SCE, and point mutation of RAS oncogene in Indian MDS patients

Thirty Indian patients diagnosed as having primary myelodysplastic syndrome as per the French-American-British classification were investigated, on admission, for the frequencies of nonrandom karyotype abnormalities, sister chromatid exchange, and point mutations of the RAS oncogene. Successful karyotype analysis was possible in 24 patients, of whom 9 (37.5%) showed nonrandom karyotypic changes. Anomalies of chromosomes 5, 7, and 8 were detected in their bone marrow (BM). In addition, two new anomalies, del(8)(q22) and +19, were observed for the first time in our series. Six MDS patients were studied for SCE in either BM or peripheral blood. These data revealed a normal SCE incidence. Of the 10 MDS patients studied for point mutations of NRAS 12 and 61 and KRAS 12 and 61, one patient exhibited a base substitution at position 1 of the 12th codon of the KRAS gene. These data, gathered for the first time on the Indian patients, throw some light on the nature of genetic changes in MDS of our country.

Genotoxicity of ‘gudakhu’, a tobacco preparation. I. In mice in vivo

‘Gudakhu’ is a paste-like tobacco preparation used widely in Orissa and neighbouring states of India. During use it is rubbed over the teeth and gum with a finger tip. Besides tobacco, it contains molasses, lime, red soil and water. The genotoxic potential of acetone extract of gudakhu was evaluated in mice in vivo using the chromosome aberration assay, micronucleus test and SCE analysis following single as well as long-term repeated treatment. The animals received an aqueous suspension of the extract via the oral route. Gudakhu extract induced significantly high frequencies, compared to controls, of chromosome aberrations, micronuclei (MN) and SCEs. Single treatment with different doses clearly revealed a distinct dose-dependent increase of the effects in all the assays. Analysis of MN in regenerated hepatocytes also indicated a significant positive correlation between time-course of chronic treatment and frequencies of micronucleated cells. But incidences of chromosome aberrations, MN and SCEs in bone marrow cells following repeated treatment for different periods did not differ greatly from each other; and these repeated treatment data, particularly in the MNT in bone marrow cells and the SCE assay, also did not differ markedly from the respective single treatment data for the same dose. This was probably due to the proliferative nature of the bone marrow cells.

Persistence of chromosomal lesions induced in vivo by mitomycin C: SCE analysis in mouse proliferating cells

Persistence of chromosomal lesions induced in vivo by mitomycin C: SCE analysis in mouse proliferating cells

Metabolic activation of indirectly acting mutagens in rat cell lines evaluated by chromosome and SCE analysis

Metabolic activation of indirectly acting mutagens in rat cell lines evaluated by chromosome and SCE analysis

Chromosomal Aberrations and Sister Chromatid Exchanges in Cultured Human Lymphocytes I. Induced by Crude Extracts of Black and Green Tea.

Crude extracts of black and green tea (BT and GT) (Camellia sp.) were assayed for chromosomal aberration and sister chromatid exchanges (SCE's) inducing effects in cultured human lymphocyte cells. The concentrations used were 0.01, 0.1, 1.0 and 10.0μg/ml. Two types of treatments were performed:(1) Type ‘A’-Cells were grown in presence of different concentrations of tea extracts.(2) Type ‘B’-Precultured (for 24 hr) cells were treated for 3 hr in plain medium (without serum, PHA and BrdU) with different concentrations of tea extracts. After two washings, cells were resuspended in complete medium (containing serum, PHA and BrdU) and incubated for desired incubation period i. e. 48 and 72 hr for chromosomal aberration and SCE analysis, respectively.The percentage values of aberrant metaphases ranged from 3.33% to 6.0% and 0.0% to 5.0% due to respective treatments ‘A’ and “B” of BT and 2.0% to 7.0% and 1.0% to 4.0% after treatments ‘A’ and ‘B’ of GT respectively. Effects did not appear to be dose dependent. Chromatid gaps and breaks were commonly observed. Terminal deletions and chromosome gaps were observed only occasionally. Treatment ‘A’ and ‘B’ of BT enhanced the SCE frequency. Treatment ‘A’ of GT enhanced the SCE's but treatment ‘B’ decreased the SCE's which might be due to the induction of DNA repair process (es). Mutagenic and antimutagenic effects of tea have been discussed. From the present study it can be suggested that while tea may protect the human genome against certain environmental agents, the possibility can not be ruled out that alone or in combination of other co-factors it may pose genetic threat.

Chromosome and SCE analysis in peripheral lymphocytes of persons occupationally exposed to cytostatic drugs handled with and without use of safety covers

The frequency of structural chromosome aberrations and sister-chromatid exchanges in peripheral blood lymphocytes of nurses handling cytostatic drugs without a safety cover is compared with that of individuals doing this work exclusively under a safety cover and with that of nurses working under similar conditions but not handling cytostatics. The mean yield of dicentric chromosomes, (4.3 ± 0.7)/1000 cells, and acentric fragments, (15.4 ± 1.4)/1000 cells, in the occupationally exposed group is significantly increased in comparison to individuals working with protection (dic: (1.1 ± 0.4)/1000 cells, ace: (11.2 ± 1.2)/1000 cells) and nurses not handling cytostatics (dic: (2.1 ± 0.5)/1000 cells, ace: (9.9 ± 1.1)/1000 cells). The frequency of chromatid breaks and SCE is not significantly different between these groups ( p > 0.05).

SCE induction and cell-cycle delay by toxaphene

Toxaphene is genotoxic in mammalian cell systems and also inhibits cell replication. It was therefore used to investigate possible masking of SCE induction due to cell-cycle delay. In this study, toxaphene-treated Chinese hamster lung (Don) cells exhibited a dose-dependent decrease in cell-cycle progression compared with untreated cells. At high, nontoxic toxaphene levels (15 μg/ml), cell cycling also slowed as the toxaphee treatment time was increased. Toxaphene induced significantly higher numbers of SCEs in treated cells, demonsstrating a dose- and treatment time-relationship. Slopes of dose-response curves were 0.29, 0.43 and 0.77 SCE/μg toxaphen for 20.5 h, 24.5 h and 28.5 h incubation, respectively. There were no changes in sCE values in control cultures even when slower dividing cells were sampled e.g. at longer incubation times. Thus, higher SCE values in Chinese hamster cells were not associated per se with slower or more delayede cells. The results demonstrate that longer toxaphene treatment times were not necessary for obtaining sufficient harlequin-stained cells for SCE analysis, but that higher numbers of SCEs occurred in slower dividing cells, following prolonged incubation of cultures treated with toxaphene.

Individual and strain differences in patterns of long-term persistence of urethane-induced sister chromatid exchanges (SCEs) in mouse lymphocytes and their relation to carcinogen susceptibility.

Using an improved microculture method, we investigated how the patterns of persistence of urethane-induced SCEs in lymphocytes differ among individuals and strains of mice (the ddY and C57BL strains), and we attempted to speculate on their relationship to carcinogen susceptibility. After a single intraperitoneal (i.p.) injection of 900 mg/kg of body weight of urethane into ten female mice each for the two strains, blood samples for the SCE analysis were collected from the tail vein at ten times during the 180 posttreatment days for each individual. Immediately after the treatment, SCE values increased to about three to four times the spontaneous values in all of the animals tested and then fell gradually. (The difference from spontaneous values was statistically significant until 120 days after the treatment.) Even after 180 days, however, some "outlier" cells with exceptionally high SCEs (greater than 20) persisted. Although there was some difference in average SCEs between the ddY and C57BL strains, the magnitude of the difference was too small to account for the difference between the strains in the incidence of urethane-induced malignancy. Also, when autopsy data at the 200th posttreatment day were matched individually with the data of SCE values within each strain, it was difficult to predict the individual risk of the occurrence of lung adenoma or other tumors from the relative difference in SCE values.

Immunogold labeling of metaphase cells

A method is presented for the phenotypic identification of metaphase cells stained for chromosome aberration and SCE analysis. The cells are labeled in suspension with antibodies conjugated with colloidal gold, and then chromosome preparations are made using a cytocentrifuge. A silver development (IGSS) procedure is used to enhance the gold labeling for light microscopy. A variety of fixatives may be employed, permitting various cytogenetic and cytochemical staining procedures to be used.

Induction of sister-chromatid exchanges in cultured human lymphocytes by Aldicarb, a carbamate pesticide

The aim of this work was to investigate the effects of Aldicarb on human lymphocytes in vitro in the presence of an exogenous metabolic activation system. This was done by means of an analysis of SCE and mitotic delay. CP was used to compare the chromosomal effects of Aldicarb with a known genotoxic agent. Our experiments showed that Aldicarb as well as CP induced a significant increase of SCE values in the absence of S9 mix. In vitro metabolic activation of both chemicals increased the SCE values. The addition of a metabolic system slightly decreased the successive mitotic progression of cells in culture.

Evaluation of genotoxic potential of vincristine from multiple parameters

The genotoxic potential of vincristine is assessed by in vivo and in vitro cytogenetic assays on two test systems using multiple parameters. In in vivo experiments it was administered intraperitoneally to Swiss albino mice at doses computed on a surface area basis to lie within the therapeutic range. The parameters employed include screening of chromosome mutational activity in bone marrow and meiotic cells for periods covering one spermatogenetic cycle, in vivo SCE analysis and sperm head abnormality assay. In vitro studies included conventional scoring for chromosome mutations from human leucocyte cultures following administration of the drug at doses within the therapeutic range on the basis of blood volume. Strict controls were employed in both test systems. The data were treated by appropriate statistical tests to evaluate their validity. The results of in vivo experiments on somatic as well as meiotic test systems reveal a lack of clastogenic action of the compound. Both sexes respond in an identical fashion regarding this activity. The SCE analysis supports the in vivo nonclastogenic action of vincristine. Total univalents significantly seen with the highest dose after 1 and 2 weeks must be considered as being important as they can lead to aneuploidy. Evidence is adduced from sperm head abnormality assay that the drug induces point mutations. The results from in vitro test system indicate a positive chromosome mutational property. This is substantiated by results of in vitro SCE analysis. A comparison of data from in vivo and in vitro test systems suggests that a metabolic conversion of the compound in the former might render it innocuous for expressing the chromosome mutational activity. They also point out that vincristine evokes a differential clastogenic response in the two test systems despite bringing about an anaphase arrest commonly in both by acting on cytokinesis.