Sister chromatid exchange in lymphocytes from patients with malignant lymphoma

Summary Sister chromatid exchange (SCE) frequencies were studied in differentially stained lymphocytes from 47 patients with malignant lymphoma. Thirteen patients were untreated when studied. The mean SCE frequency [±standard error (SE)] for these patients was 12.7±0.9 per mitosis. The mean score for 40 controls was 6.1±0.3. SCE mean scores were significantly higher in the untreated patients than in the controls (P<0.001). Seven patients were treated with radiotherapy alone. They demonstrated a mean SCE frequency (8.8±0.8) significantly lower (P<0.01) than that found in untreated patients. Eleven patients received cyclophosphamide within 4 weeks prior to study. They demonstrated a mean SCE frequency (14.3±1.3) significantly higher (P<0.05) than that found in patients who had received regimens that did not contain cyclophosphamide in the prior 4 weeks (11.1±1.3) or who had been off drugs for at least 8 weeks (10.1±0.8). Our data suggest that untreated patients with malignant lymphoma have elevated SCE frequencies, which may be further increased by certain chemotherapeutic agents.     

Sister chromatid exchange in murine alveolar macrophages,regenerating liver and bone marrow cells — a simultaneous multicellular in vivo assay

Differential labeling of sister chromatids was achieved simultaneously in murine alveolar macrophages, regenerating liver, and bone marrow cells of partially hepatectomized mice as well as in alveolar macrophages and bone marrow cells of nonhepatectomized mice. The mean frequency of SCE/cell ±S.D. and the percentage of second division cells for each cell type were determined. No significant differences in mean frequencies of SCE/cell were observed among the cell types or between hepatectomized (alveolar macrophages –3.6±2.2, bone marrow –3.4±2.2; regenerating liver –3.6±2.4) and nonhepatectomized (alveolar macrophages —3.4 ±1.9; bone marrow —2.9±1.8). Although the percentage of second division cells was dependent upon cell type, no significant differences were apparent between hepatectomized (alveolar macrophages —57±8%; bone marrow —37±6%; regenerating liver –65±6%) and nonhepatectomized mice (alveolar macrophages –53±6%; bone marrow –36±4%). Comparisons between BrdU treated and nontreated nonhepatectomized mice revealed no significant alteration in mitotic yields.     

Sister chromatid exchange in lymphocytes from patients with Acute lymphoblastic leukemia

Summary Sister chromatid exchange (SCE) frequencies were studied in peripheral lymphocytes from 16 patients with newly diagnosed acute lymphoblastic leukemia (ALL) prior to the initiation of chemotherapy. The mean SCE frequency ( ±SE) for these patients was 12.2±0.2 per metaphase, which was significantly higher (P(0.001) than the mean SCE score for 14 agematched controls, 7.6±0.2. Five of these patients were studied again while they were receiving maintenance therapy consisting primarily of daily 6-mercaptopurine and weekly methotrexate. Their remission SCE levels remained significantly higher than controls (P(0.005). In addition, SCE levels were studied in 7 long-term survivors of ALL. Three of these patients had been receiving continuous maintenance therapy for at least 3 years. Their mean SCE scores were significantly greater than controls (P(0.005). The other 4 patients had finished their final course of chemotherapy at least 8 months prior to the time of sampling, and their mean SCE scores were not significantly different from controls (P>0.10). These data indicate that untreated patients with ALL have increased SCE levels which remain elevated during periods of remission maintained with chemotherapy. However, longterm survivors of ALL who are in remission and off chemotherapy do not demonstrate significantly increased SCE frequencies.     

Sister chromatid exchanges in leukocytes of patients with cancer of cervix uteri

Summary The frequency of sister chromatid exchange (SCE) was investigated in 13 women with cervical cancer together with 11 control women. The SCE frequencies were found to be 10.05±2.35 and 6.95±1.53 in cancer cases and controls, respectively. The SCE values of cancer cases deviate significantly from that of controls. The SCE in chromosome groups E, F, and G was found to be more in comparison to controls (P<0.001). This preliminary study indicates the possibility of using SCE as a preclinical marker.     

Induction of sister-chromatid exchanges by DNA-damaging agents and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in synchronous Chinese hamster ovary (CHO) cells

Synchronous CHO cells were obtained by mitotic selection; synchrony was maintained up to the 5th cell cycle. The mitotic cells were seeded into T-25 flasks or P-60 plastic petri dishes, and cultured for 1 h at 37 degrees C, then the cells were treated by X-ray, UV light, and mitomycin C. The cells were then cultured for 2 cell cycles with TPA and BrdUrd and sister-chromatid exchanges (SCE) analyzed by the FPG method. Following X-irradiation, the frequency of induced SCE increased linearly with dose reaching a maximum of 19.8 times the control frequency after 200 rad. With higher doses, the SCE frequency declined. In the presence of TPA, SCE frequencies were 1.8 times control levels for all X-ray doses studied (0-800 rad), the frequency seen in non-irradiated cultures treated with TPA. The induced SCE frequency also increased linearly following treatment with UVL and mitomycin C, reaching levels higher than 1.8 times controls with doses exceeding 2.5 J/m2 UVL or mitomycin C (30 min). In the presence of TPA, the SCE frequencies increased to 1.8 times controls following low UVL and mitomycin C doses, but were not influenced by TPA in the higher dose range (above 2.5 J/m2 or 10(-10) M mitomycin C. Most of the SCE were induced by X-rays during the first S phase after treatment. Following higher UVL doses (5 J/m2), however, the SCE frequency remained elevated (1.5 times controls) for 4 cell cycles after exposure.     

Sister chromatid exchanges in patients with precancerous and cancerous lesions of cervix uteri

Summary The frequency of sister chromatid exchanges (SCEs) was studied in leucocytes from 46 patients with cervical carcinoma, 89 precancerous lesions, and 43 age-matched control women. The frequency of SCEs was found to be 10.15 ±2.49 in cancer, 8.83±2.15 in precancerous lesions, and 7.55±2.24 in controls. The analyses of SCE data revealed a highly significant (P<0.001) increase in precancerous and cancerous lesions compared to controls. The intra-chromosomal distribution of SCEs revealed a random increase in various chromosomal groups in patients with cancer and dysplasia compared to controls. The mean SCE level among various groups of precancerous lesions according to severity of pathological condition did not show significant differences. However, 70.8% of dysplasia cases revealed SCE levels higher than the average in controls. The increased frequencies of SCEs in the majority of cancer patients and a few, precancerous lesions indicate that individuals with high SCE levels may be at a high risk of developing cancer. Thus the usefulness of SCE levels as a preclinical marker to identify the high risk group of dysplasias needs to be ascertained by follow-up studies; these are in progress.     

Changes of common fragile sites on chromosomes according to the menstrual cycle

Summary The frequencies of chromosomal breaks and sister chromatid exchanges (SCE) are influenced by pregnancy, oral hormonal contraceptives and the menstrual cycle. The changes in the number and sites of spontaneous and aphidicolin-induced breaks on chromosomes from peripheral blood lymphocytes during the menstrual cycle were examined in 8 healthy women. Menstrual cycle was determined by menstruation and the quantity of serum estrogen, progesterone and luteinizing hormone. The number of spontaneous breaks at the follicular phase, the interval phase (which includes ovulation) and the luteal phase were 3.1 ± 1.1, 2.7 ± 2.3 and 3.9 ± 2.6 per 100 mitoses, respectively. The frequencies of aphidicolin-induced breaks in the same phases were 95.8 ± 23.3, 90.6 ± 14.3 and 122.7 ± 20.1 per 100 mitoses, respectively. The higher frequency at the luteal phase was statistically significant compared with the other phases. In the luteal phase, bands 2q32, 3q27, 6q26 and 16q23 had higher frequencies of breaks (P < 0.05); however, breaks at band 9q32 decreased significantly. SCE showed considerable variation, but with no statistical significance.     

Effect of tissue culture variables on sister chromatid exchange in a nontransformed rat cell line

Summary The frequency of sister chromatid exchange (SCE) was determined in a nontransformed diploid rat cell line, FR3T3, under several tissue culture variables such as cultivation temperature, growth conditions of cells, and concentrations of 5-bromo-2′-deoxyuridine (BrdU). The conclusions to be drawn from these experiments are: (a) The cell growth and mechanism(s) of SCE formation in FR3T3 cells are largely temperature independent (or efficiently regulated) in the range between 33 and 40.5°C. (b) The concentration limits for BrdU incorporation are 5 to 100 μM; baseline frequency is about 11 SCE/metaphase (constant up to 20 μM BrdU) and increases only moderately at higher BrdU concentrations. (c) Toxic levels of BrdU (150 μM) cause a decrease of SCE rates below that found at 100 μM, presumably due to selective cell death. (d) Keeping cells growth arrested over a long period causes substantial SCE induction after replating. (e) Induced increase of SCEs probably occurs in this manner during the first cell cycle after release from growth arrest. It is no longer detectable after the fourth consecutive cell division. This work was supported by a grant from the Medizinisch-wissenschaftlicher Fond des Bürgermeisters der Bundeshauptstadt Wien.     

In vivo sister chromatid exchange in cells of various organs of the mouse

In vivo sister chromatid exchange (SCE) in mouse cells derived from various organs was studied by infusing BrdU from the tail vein. It was found that at BrdU concentrations ranging from 2.2–13.5 g/g/h, the SCE frequency in bone marrow cells seemed to stay at a constant level (1.5–2/cell/two cell cycles) whereas it started to rise as the BrdU dose exceeded this dose range. When BrdU within this dose range was infused continuously from the tail vein for appropriate hours to label chromosomes in various organs, the average SCE frequencies per cell were found to be 1.64 in bone marrow cells, 1.82 in spermatogonia, 1.99 in splenic cells, 2.89 in intestinal cells and 3.69 in cells from adjuvant stimulated lymph nodes. It is suggested that the spontaneous level of the in vivo SCE frequency might be about 1.5–2/cell/two cell cycles in the mouse. In cells derived from intestine and adjuvant stimulated lymph node, some unknown factors might work as a inducer of SCEs resulting in a significant increase in the SCE frequency in these organs.     

Cytogenetic analysis in workers occupationally exposed to nickel carbonyl

Chromosomal aberration and sister-chromatid exchange (SCE) base-line frequencies and SCE frequencies induced by 10 ng/ml mitomycin C (MMC) were analysed in cultured peripheral lymphocytes of 65 workers occupationally exposed to nickel carbonyl Ni(CO)4. The subjects were divided into 4 groups: (1) control; (2) exposed to nickel carbonyl (= exposed); (3) cigarette smokers; (4) smoking-exposed. The results show that there are no significant differences in chromosomal aberration frequencies, breaks or gaps, between the various groups. However, the SCE base-line frequency of the smoking-exposed group, with an average of 7.7/cell, was significantly higher than that of the control group, with an average of 6.5/cell (P less than 0.01), and also than that of the exposed group with an average of 5.9/cell (P less than 0.01). Similarly, the SCE frequency induced by 10 ng/ml MMC in the smoking-exposed group which averaged 15.5/cell was significantly higher than that of the control group (average of 13.2/cell (P less than 0.05], and also than that of the exposed group with an average of 12.3/cell (P less than 0.01). Under our experimental conditions, it may be that the level of exposure was not high enough to elicit an increase in chromosomal aberrations and SCE frequencies in the non-smoker exposed group. The fact that an increase in SCE frequencies was only found in the smoking-exposed group implies that the two factors, smoking and exposure to nickel carbonyl, are jointly responsible for the result.     

Cell-cycle duration and sister-chromatid exchange frequency in cultured human lymphocytes

Whole heparinized blood samples from normal human donors were grown in culture media containing 10 μg/ml of bromodeoxyuridine. Lymphocytes were harvested after 58, 70, 72 and 80 h and scored for sister-chromatid exchanges (SCEs) under a fluorescence microscope. SCEs which occured during the first and second cell cycles were counted in second or third generation cells selected on the basis of their chromosome fluorescence patterns. The results of a preliminary study showed the mean SCE frequency per cell at 72 h to be 9.0 for second generation cells and 7.8 in third generation cells (P < 0.01). A second study, using culture medium with heat-inactivated fetal-calf serum, gave similar results (9.4 vs. 7.8, P 0.001) at 70 h. Therefore, the difference in SCE frequency between second and third generation cells at 70 or 72 h cannot be attributed to heat-labile substances of serum origin. An additional finding in the second study was that SCE frequencies in third division cells at 70 and80 h were the samee as those of second division cells at 58 h but significantly less (P < 0.001) than the frequency in second division cells at 70 h. These data were interpreted as arising from at least two different lymphocyte populations; one group of cells that is either slower growing or slower in phytohemagglutinin stimulation, with a higher SCE frequency which does not reach second division until 70 or 80 h, and a more rapidly dividing (or more quickly stimulated by phytohemagglutinin) population with a lower SCE frequency which reaches second division at 58 h and third division by 70–80 h. Whether or not this hypothesis is correct, the data show that SCE frequency varies significantly with cell-cycle duration. Since some carcinogens have been shown to alter cell kinetics (Craig-Holmes and Shaw, Mutation Res. 46 (1977) 375), changes in SCE frequency which are caused by a change in cell kinetics must be considered a factor in determining the mutagenicity of an agent by its ability to increase SCE frequency.     

Chromosomal Aberrations (CA), Sister Chromatid Exchanges (SCE), and High-Frequency Cells (HFC) Frequencies in Peripheral Blood Lymphocytes of Tunisian Gasoline Truck Loaders

Gasoline constitutes a mixture of chemicals that contain well-known genotoxicants. Thus, chronic occupational exposure to gasoline may be considered to possess genotoxic risk. In this study, the frequencies of total chromosomal aberrations (TCA), aberrant cells (Ab.c.), sister chromatid exchanges (SCE), high-frequency cells (HFC), and high-frequency cell individual (HFI) were investigated in peripheral blood lymphocytes from 17 gasoline-exposed workers (10 smokers and 7 non-smokers) and 22 unexposed reference subjects (12 smokers and 10 non-smokers). The exposed subjects were gasoline truck loaders at a gasoline company from Tunis City, north of Tunisia. The results indicate multiple CA, such as dicentrics (DIC), chromatid breaks (SB), and chromosome breaks (DB). A significant difference was observed in TCA and Ab.c. frequencies between exposed and unexposed groups (p < 0.01). A significant difference was found in frequencies of SCE (p < 0.01) and HFI (p < 0.05) between exposed and unexposed groups. SCE and TCA frequencies of smokers were found to be significantly higher than those of non-smokers in both groups. There was an interaction between gasoline exposure and smoking habit for TCA (p = 0.020), but not for SCE. Our findings indicate that gasoline truck loaders were under risk of significant cytogenetic damage that was enhanced by their smoking habit.     

Apolipoprotein E phenotypes in patients with myocardial infarction

Summary The frequencies of genetic apo E isoforms E2, E3 and E4 were determined in 523 patients with myocardial infarction and compared to those in a control group (1031 blood donors). A significant difference in the frequency of apo E4 was noted between patients and controls (0.05> P>0.025). No differences in the frequencies of isoforms E3 and E2 were observed. In particular, there was no significant difference between the two groups in the frequency of apo E2 homozygosity. a condition that is associated with type III hyperlipoproteinemia. However, all E2 homozygote survivors of myocardial infarction had hyperlipoproteinemia type III (cholesterol 269±29 mg/dl; triglyceride 419±150 mg/dl; age 54±14 years; N=5). On the contrary, E2 homozygote controls (all apo E-2/2 blood donors and their apo E-2/2 relatives who were from the same age range as the patients) had primary dysbetalipoproteinemia but normal or subnormal plasma cholesterol concentrations (cholesterol 184±28 mg/dl; triglyceride 151±52 mg/dl; age 56±13 years; N=11). This indicates that E2 homozygotes with hyperlipoproteinemia type III who occur rarely in the population but comprise about 1% of myocardial infarction patients have a markedly increase risk for coronary atherosclerosis, whereas the risk for E2 homozygotes with normal or subnormal plasma cholesterol (=primary dysbetalipoproteinemia) may be considerably lower than for the general population. The data illustrate the complex relationship between apo E genes, lipid levels, and risk for atherosclerosis.     

Paradoxical changes in SCE frequencies persistently elevated in vivo, on exposure to a mutagen in vitro

Lymphocyte cultures from 4 individuals with persistently significantly elevated frequencies of sister-chromatid exchange (SCE) were examined with no treatment, and with 2 concentrations of mitomycin C. In each of the 4 cases, the mean level of SCEs in the untreated lymphocytes exhibited a paradoxical reduction in SCE frequency when exposed to the lower (0.005 microgram/ml) of the two doses of mitomycin C. At the second higher dose of mitomycin C (0.025 microgram/ml) the mean level of SCE/cell exceeded the untreated mean. When the distributions of SCE/cell were examined it appeared that the untreated cultures had two or more populations of cells; one was in the normal SCE frequency range, while the second population was in an elevated SCE frequency range. The paradoxical reduction in SCE frequency was apparently due to elimination of, or mitotic inhibition of cells in the highest range of SCE frequency, while a small elevation in SCEs was initiated in the cells with a normal SCE frequency. Thus, mean levels of SCE/cell can be misleading. This data suggests that new exposure to the same or a different genotoxic agent might possibly result in a misleading lowering of the mean SCE frequency.     

Induction of sister chromatid exchanges in human cells by fluorescent light.

The Brd-U differential staining technique was utilized to examine the induction of sister-chromatid exchanges (SCE) by fluorescent ligt in human fetal lung fibroblasts (IMR-90). Exposure of these cells in media to fluorescent light resulted in an increase in SCE frequencies from a background level of 8.5 SCE/cell to 20.5 SCE/cell. Cellular replication kinetics were also inhibited by fluorescent light exposure. Exposure of cells to fluorescent light in phosphate buffered saline (PBS) resulted in a two-fold increase in SCE levels and incresed inhibition of cell replication, indicating that culture media may have a protective effect. Determinations of SCE frequencies with blocking filters indicated that the fluorescent light wavelengths responsible for SCE induction were in the near-ultraviolet spectrum between 300 and 390 nm. Culturing cell sin media that had been exposed to fluorescent light resulted in a significant increase in SCE levels, 14.5 ± 1.5 vs. 7.5 ± 0.65, demonstrating the contribution of media photoproducts to SCE induction. The role of media photoproducts was further reinforced by finding a significant decline in fluorescent light induced SCE in cells cultured in medium deficient in three known photosensitizers (phenol red, tetracycline and riboflavin) for 2–3 weeks prior to exposure.Since SCE have been shown to be a sensitive indicator of DNA damage, these results indicate that fluorescent light can induce genetic damage in human cells. These findings are also of importance to investigators culturing cells in laboratories with fluorescent illumination.     

人脐带间充质干细胞条件培养基联合白藜芦醇对人滋养层细胞的作用

目的:探讨人脐带间充质干细胞条件培养基联合白藜芦醇对人绒毛膜外滋养层细胞凋亡的影响。方法:通过CCK8细胞活力检测试剂盒测定白藜芦醇及其与人脐带间充质干细胞条件培养基共同处理人绒毛膜外滋养层细胞HTR8后对细胞增殖及活性的影响;细胞迁移试验检测白藜芦醇和人脐带间充质干细胞条件培养基对细胞迁移能力的影响;显微镜观察细胞形态,并用流式细胞仪检测细胞凋亡率的变化;Western blot检测白藜芦醇和人脐带间充质干细胞条件培养基对细胞凋亡相关蛋白Bax、Bcl-2以及迁移相关蛋白MMP-9表达的影响。结果:白藜芦醇能够抑制HTR8细胞增殖,抑制细胞迁移及MMP-9蛋白的表达,改变Bax和Bcl-2蛋白表达诱导细胞凋亡的作用。而人脐带间充质干细胞条件培养基能够逆转白藜芦醇对细胞的抑制作用。结论:人脐带间充质干细胞条件培养基能够通过调控Bax、Bcl-2、MMP-9的蛋白表达逆转白藜芦醇对人绒毛膜外滋养层细胞的抑制作用。人脐带间充质干细胞条件培养基可作为潜在的治疗人绒毛膜外滋养层细胞功能障碍的临床手段,孕妇需要小心使用白藜芦醇。     

Sister chromatid exchanges in cultured immature embryos of wheat species and regenerants

Immature embryos of Triticum aestivum (ten cultivars and lines), T. durum, T. dicoccum and T. monococcum were cultured in vitro on MS medium supplemented with 1 or 2 mg/l of 2,4-D and 20 or 30 g/l of sucrose for 3 days and processed to score sister chromatid exchanges (SCEs) per chromosome. Media components affect DNA replication from the start of the culture. The SCE frequencies were dependent on the genotype and were not correlated with the degree of ploidy. They increased after doubling of the concentration of 2,4-D and/or sucrose, except in one cultivar of T. aestivum. The mean numbers were lower than observed in root meristems of T. aestivum (two cultivars) and T. dicoccum. Immature embryos of regenerants of T. aestivum (one cultivar) and T. durum demonstrated variable SCE frequencies, which may have been caused by mutations in the parental cell cultures. In the T. aestivum embryos the lowest frequencies were found in regenerants obtained from explants with the highest frequencies.     

Cell cycle studies in chorionic villi

Summary We have studied the cell cycle of cells obtained from chorionic villi in direct and culture preparations by incorporation of the thymidine analogue BrdU to produce latelabelling or sister chromatid differentiation patterns. We have, therefore, been able to estimate the duration of the cell cycle and, more specifically, the length of some of its phases. While results for chorionic villus sample cells in culture resembled those obtained for fibroblasts, data for the spontaneously dividing trophoblastic cells in direct preparations were different. Villi exposed to BrdU immediately after sampling showed a slight delay in the incorporation of the analogue and a lower percentage of labelled cells compared to villi treated after an overnight incubation, probably due to a temporary effect of the sampling technique. Results from semi-direct protocols suggest that cells have a G2 of no more than 4h, and a mid-S phase of 10–16h. The G1 period is very variable. After 48 h incubation with BrdU, only 4% of cells reach their second generation, whereas this percentage increases up to 70% after 72h, indicating that under these experimental conditions most cells have a cell cycle of approximately 36 h. The average number of sister chromatid exchanges was similar in both direct preparations and cultures: 5.2±2.1 SCE per cell.     

Frequencies of sister-chromatid exchanges in relation to cell kinetics in lymphocyte cultures

The frequency of sister-chromatid exchanges (SCE) was determined on second-division metaphase of lymphocytes stimulated by phytohaemagglutinin (PHA) during 9 days of culture.

By using either a continuous or a pulsed bromodeoxyuridine (BUdR) treatment, cells were selected that had divided only twice, or at least twice, after different culture periods. No significant differences were observed in the SCE frequencies among the various samples. The incidence of SCE appears to be independent of the proliferation properties of cultured lymphocytes, such as length of cell cycle, fast or delayed response to PHA and number of divisions performed in vitro.     

The influence of 2,4-dichlorophenoxyacetic acid on cell cycle Kinetics and Sister-Chromatid Exchange Frequency in Garlic (Allium sativum L.) Meristem Cells

The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) at low concentrations on cell cycle duration and sister-chromatid exchange (SCE) frequency was studied using meristem root-tip cells ofAllium sativum L. 2,4-D induced a marked prolongation of the cell cycle. At the same time, small but statistically significant increases in SCE frequencies were observed at 5 μM and 15 μM 2,4-D concentrations. The significance of these findings in the evaluation of mutagenic activity of 2,4-D is discussed.