Non-uniform distribution of sister chromatid exchanges in human lymphocytes

To test whether sister chromatid exchange (SCE) scores on human chromosomes have a uniform distribution, simulated SCE scores were generated and compared with observed scores using log-linear models. The analysis was performed at the level of the chromosome groups. Using this method we first tested whether the number of SCEs was distributed uniformly, i.e. proportional to the relative length of the chromosomes. Refinements of this hypothesis were made by considering a variable region around a first SCE to be inert for other SCEs and by making the occurrence of an SCE on a chromosome dependent on the occurrence of another SCE on the same chromosome. In further analyses it was tested whether the number of SCEs was proportional to the number of G bands on a chromosome, or to the DNA content of the chromosomes. None of the tested hypotheses fitted the observed data, establishing the non-uniform distribution of these events.     

Variation in the frequency of sister chromatid exchanges in repeated human lymphocyte cultures

Summary Repeated blood samples from two healthy donors were taken over a period of about one year to determine the temporal variation in human lymphocyte baseline sister chromatid exchange (SCE)-frequencies. The investigations were performed on whole blood cultures and purified lymphocyte cultures using a standardized protocol for blood collection and cultures. Significant differences in the frequencies of SCEs were found between the two cultivation systems and the two blood donors but also between repeated cultures of the same individual. There was no systematic relationship between the proliferation of the cultures and the basal SCE values. The results indicate the necessity of concurrent controls and repeated blood samples whenever SCEs are used as a test for monitoring human exposure to potential mutagens. Temporal variation in human lymphocyte baseline SCE frequencies is a limiting factor for the detection of minor effects of genotoxic agents.     

Modulation by novobiocin of sister-chromatid exchanges induced by tumor necrosis factor in human lymphocytes.

The effect of the antibiotic novobiocin on human recombinant tumor necrosis factor (TNF)-induced sister-chromatid exchanges (SCEs) were examined in human peripheral blood lymphocytes. TNF, when introduced in a dose range of 10-1000 U/ml at the initiation of culture, was found to cause a significant increase in SCE frequency. The simultaneous addition of TNF and novobiocin (25 micrograms/ml) in the assay resulted in no increase of SCE frequency. Delayed (for 24 h) addition of novobiocin suppressed the induction of SCEs by 50, 100 and 500 U/ml but not by 1000 U/ml of TNF.     

Both cross-links and monoadducts induced in DNA by psoralens can lead to sister chromatid exchange formation

The relative importance of DNA-DNA cross-links and bulky monoadducts in sister chromatid exchange (SCE) formation was investigated in three human fibroblast cell lines with different repair capabilities. These cell lines included normal cells, which can repair both classes of lesions; xeroderma pigmentosum (XP) cells, which cannot repair either psoralen-induced cross-links or monoadducts; and an XP revertant that repairs only cross-links and not monoadducts. SCEs were induced by two psoralen derivatives, 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and 5-methylisopsoralen (5-MIP). After activation with long-wave ultraviolet light, HMT produces cross-links and monoadducts in DNA, whereas 5-MIP produces only monoadducts. In normal human cells both psoralens induced SCEs, but if cells were allowed to repair for 18 h before bromodeoxyuridine (BrdUrd) was added for SCE analysis, the SCE frequency was significantly reduced. XP cells showed an SCE frequency that remained high regardless of whether SCEs were analyzed immediately after psoralen exposure or 18 h later. In the XP revertant that repairs only cross-links, both psoralens induced a high yield of SCEs when BrdUrd was added immediately after psoralen treatment. When XP revertant cells were allowed 18 h to repair before addition of BrdUrd, the SCEs induced by HMT were greatly reduced, whereas those induced by 5-MIP were only slightly reduced. These observations indicate that both cross-links and monoadducts are lesions in DNA that can lead to SCE formation.     

The influence of GSTM1 and GSTT1 genotypes on the induction of sister chromatid exchanges and chromosome aberrations by 1,2:3,4-diepoxybutane

Cytogenetic tests - chromosome aberrations (CA), sister chromatid exchanges (SCE) and micronuclei (MN) - are most often applied in biomonitoring of the genotoxicity of potentially carcinogenic chemicals in human cells. One of the extensively studied genotoxins is diepoxybutane (DEB) - reactive biometabolite of butadiene (BD). Several studies showed a high SCE induction in human lymphocytes exposed in vitro to various concentrations of DEB. DEB also proved to be a potent inducer of chromosome aberrations and micronuclei. A bimodal distribution of SCE frequency after in vitro DEB treatment was observed. The aim of the present study was to examine the ability of DEB to induce different individual cytogenetic response measured by SCE and CA frequency. The possible influence of genetic polymorphism has also been taken into account, by including donors representing positive or null GSTM1 and GSTT1 genotypes. Our study supported the earlier results showing that DEB is an effective inducer of SCEs and CAs, causing also the decrease in replication index (RI). DEB bioactivity measured by SCE induction - but not by CA test - was significantly higher in GSTT1 negative than in GSTT1 positive donors. GSTM1 polymorphism had no influence on these endpoints. The donors GSTT1-/GSTM1+ were shown to be slightly more sensitive to DEB than GSTT1-/GSTM1- individuals. There was also observed a unimodal distribution of DEB-induced SCEs and CAs in the group, despite the fact that the experiment was performed on the lymphocytes obtained from both GSTT1 positive and negative donors.     

Effects of cell fusion and deoxynucleosides on sister-chromatid exchanges in B-lymphoblastoid cell lines from 5 Bloom syndrome patients

The effect of cell fusion and deoxynucleosides (deoxyadenosine, dA; deoxyguanosine, dG; deoxycytidine, dC; thymidine, T) on sister-chromatid exchanges (SCEs) in Bloom syndrome (BS) was studied in two types of BrdU (bromodeoxyuridine)-sensitive and BrdU-resistant B-lymphoblastoid cell lines (LCLs) with respect to cellular proliferation in BrdU-labeled culture conditions. Cell fusion between BrdU-sensitive and BrdU-resistant BS B-LCLs did not exhibit complementation, although when any of the BS B-LCLs (retaining high SCE character) labeled with BrdU were fused with non-labeled normal cells, the hybrid cells had a normal level of SCE at the first mitosis after fusion. Deoxycytidine addition showed no effect on SCEs in normal cells but decreased SCEs in BS cells from the baseline level of 70 SCEs/cell to about 60 SCE/cell. Purine deoxyribonucleosides (dG and dA) caused a significant concentration-dependent increase in SCE frequency both in normal and BS cells. Although T caused a 2-fold increase in normal SCEs, it highly decreased BS SCE from 70 SCEs/cell to 35 SCEs/cell. FrdU did not greatly affect BS SCE in the presence of BrdU and T. These observations indicate strongly that BS cells may have a low thymidine pool compared with normal cells, which could account for a more efficient BrdU substitution in the DNA thus potentiating the template effect on SCE.     

Sister-chromatid exchange in human B- and T-lymphocytes exposed to bleomycin, cyclophosphamide, and ethyl methanesulfonate.

Sister-chromatid exchange (SCE) frequencies were investigated in mitogen-stimulated cultures of highly purified human peripheral blood B- and T-lymphocytes exposed to bleomycin (BM), cyclophosphamide (CP), or ethyl methanesulfonate (EMS). In untreated controls, T-lymphocytes showed twice as many SCEs as B-lymphocytes. CP (with metabolic activation) and EMS significantly increased the SCE frequencies. EMS induced a similar, dose-dependent SCE increase in both cell populations, whereas CP induced more SCEs in T- than in B-lymphocytes. No clear SCE increase was found in B- and T-lymphocytes treated with BM.     

A comparison of SCE test in human lymphocytes and Vicia faba: a hopeful technique using plants to detect mutagens and carcinogens

A comparison of the Vicia faba SCE with the human lymphocyte SCE test was made with regard to their capacity to detect mutagens. Twelve chemical agents that can induce sister-chromatid exchanges (SCEs) in both Vicia faba root tip cells and human lymphocytes were used. The results indicate that the plant SCE technique is a sensitive test for screening various chemical agents including food preservatives, pesticides and essences which may have mutagenic activity. A new simplified procedure was introduced for differential staining of sister chromatids in Vicia faba.     

小鼠体内脾脏,骨髓及精原细胞姐妹染色单体交换的比较研究

本文对几种化学诱变剂诱发小鼠体内脾脏、骨髓和精原细胞的SCE进行了比较研究,同时分析了几类常见化合物在小鼠脾脏细胞中诱发SCE的活力。结果显示诱变剂在脾脏细胞中诱发SCE比骨髓和精原细胞敏感。几类化合物都能显著地诱发小鼠脾脏SCE的增加,与对照相比差异显著(P<0.05)或极显著(P<0.01),说明利用小鼠脾脏细胞检测环境诱变物是相当灵敏的。     

Genotoxic effects induced by fotemustine and vinorelbine in human lymphocytes

The aim of this study was to investigate the in vitro genotoxic effects of the anticancer drugs fotemustine and vinorelbine on human lymphocytes and to determine individual and sex-related responses to these drugs. Fotemustine is a DNA-alkylating drug while vinorelbine is a semi-synthetic Vinca alkaloid. The study was carried out with twenty independent healthy donors for each drug. We have tested the ability of these drugs to induce chromosome aberrations (CAs) and sister chromatid exchanges (SCEs) as well as effect on the mitotic index (MI) in cultured human lymphocytes. Fotemustine was shown to induce CAs and SCEs at all concentrations tested (2, 4 and 8 microg/ml) in a dose-dependent manner. Additionally it also decreased the mitotic index in a similar dose-dependent manner. Vinorelbine had no effect on structural CAs, but it significantly increased the numerical CAs at all doses tested (0.5, 1 and 2 microg/ml). Vinorelbine also induced SCE events and increased the MI values. Two-way analyses of variance were used to compare the individual and gender-related susceptibilities to fotemustine and vinorelbine with respect to the CA, SCE and MI values. The results indicated that individuals in fotemustine treatment groups showed different genotoxic responses with respect to CA and SCE induction and additional findings indicated a gender-specific response in this group. Individuals in the vinorelbine test group also exhibited statistically significant numerical CA, SCE and MI responses to vinorelbine. A statistically significant gender-related SCE response to this drug was also evident. This study indicates that these drugs have potentially harmful effects on human health.     

Erythrocytes modulate the baseline frequency of sister-chromatid exchanges and the kinetics of lymphocyte division in culture

The baseline sister-chromatid exchange (SCE) frequencies of human plasma lymphocyte cultures (PLC), but not pig PLC, were nearly twice as high as those of whole-blood cultures (WBC). Addition of human red blood cells (RBCs) to human PLC decreased the SCE frequency in proportion to the RBC-leukocyte co-incubation interval. When the period of RBC-leukocyte co-incubation was equivalent to the total length of the culture period (72 h), the SCE frequency was similar to that observed in WBC. Shorter co-incubation periods yielded SCE frequencies intermediate between those of PLC and WBC. Regardless of the species, cell proliferation was slower in PLC than in WBC. Experiments where RBCs were added to PLC showed that the time sequence of RBC incorporation also affects the cell-cycle progression of human and pig lymphocytes. When either human or pig RBCs were added immediately after PLC stimulation, the cell-cycle kinetics was similar to that of WBC. Shorter co-incubation periods made cell-cycle progression intermediate between PLC and WBC values. Thus, PBCs modulate the baseline frequency of SCEs in human PLC and the cell-cycle progression of both human and pig lymphocytes in a time-dependent manner. Two possible hypotheses for the heightened frequency of SCEs of human lymphocytes in RBC-free cultures were assessed. The loss of RBC-to-lymphocyte cellular contact in PLC did not influence the SCE frequencies of lymphocytes. Finally, the increase of SCEs in human PLC could not be related to differences in the generation time of lymphocytes in culture.     

Induction of sister-chromatid exchanges by 2-aminofluorene in cultured human lymphocytes with and without erythrocytes.

2-Aminofluorene (2-AF), an indirect mutagen reported to be metabolically activated by erythrocytes in the Salmonella mutagenicity test, was studied for the induction of sister-chromatid exchanges (SCEs) in human lymphocytes in vitro with (whole-blood cultures) and without erythrocytes (isolated lymphocyte cultures). 2-AF (0.025-0.8 mM) was present in the cultures for the last 48 h of 72-h cultures. In both types of culture, SCEs increased in a dose-dependent manner, with a statistically significant elevation already at the lowest concentration of 2-AF tested and maximum responses of 2.4-fold (whole blood) and 2.1-fold (isolated lymphocytes), in comparison with mean SCEs/cell in control cultures, at 0.4 and 0.2 mM concentrations (respectively). Thus, the induction of SCEs by 2-AF was not dependent on the presence of erythrocytes. Styrene (2 mM), a positive control chemical known to require erythrocytes for efficient SCE induction in vitro, was shown to produce a 4.9-fold increase in SCEs in whole-blood cultures, but only a slight (1.3-fold) effect in isolated lymphocyte cultures. The results suggest that leukocytes, but not erythrocytes, are important in the metabolic activation of 2-AF in the human lymphocyte SCE assay.     

Effects of 5-bromo-2-deoxyuridine concentration and alpha-naphthoflavone on the association between smoking and the frequency of sister-chromatid exchanges in lymphocytes from maternal and cord blood

The frequency of sister-chromatid exchanges was analyzed in maternal and cord blood lymphocytes obtained at delivery from 23 nonsmokers and 21 smokers. Lymphocytes were cultured under 3 conditions: in the presence of 100 microM 5-bromo-2-deoxyuridine (BUdR), 20 microM BUdR and 20 microM BUdR with 40 microM alpha-naphthoflavone (ANF). Under all assay conditions, frequencies of SCEs were consistently higher for maternal lymphocytes than for cord lymphocytes. There was no association between SCE values for cultures of the same blood specimen with 100 microM BUdR and 20 microM BUdR. When cultured with 100 microM BUdR, maternal lymphocytes from smokers had a mean SCE frequency of 13.5, which was significantly higher than the value of 11.1 observed for nonsmokers (p = 0.001 by the Wilcoxon rank sum test). Maternal smoking had no significant effect on overall frequencies of SCEs in maternal blood cultured with 20 microM BUdR either with or without ANF or when the differential between cells cultured with and without ANF was considered. Use of caffeinated beverages was associated with increased SCE values for maternal lymphocytes cultured with 20 microM BUdR (Tau beta = 0.36, p = 0.02 for the Kendall's Rank Correlation), but no such association was seen with 100 microM BUdR. For cord blood lymphocytes, however, neither smoking nor caffeine use were associated with SCE values obtained by any of the assay conditions used. The findings suggest that results of human monitoring studies using SCEs could differ depending on the concentration of BUdR used in cultures.     

The effect of chlorpromazine on SCE frequency in human chromosomes: An in vitro and in vivo study

Schizophrenic patients who were receiving, or who had received chlorpromazine showed SCE levels similar to those in a normal control population. Of 8 normal individuals whose lymphocytes were exposed in vitro to chlorpromazine (0.05–2.00 μg/ml) for two cell cycles, 4 showed a significant increase in SCE, 3 showed no increase and 1 a decrease compared with untreated lymphocytes. Lymphocytes from a further 8 donors treated with 2.0 μg/ml chlorpromazine prior to mitogen stimulation (G₀ lymphocytes) showed a similar SCE response. Only 3 of the 8 donors showed a significant increase in SCEs over the baseline level. When proliferating lymphocytes were exposed to chlorpromazine 38 h after culture initiation and prior to the addition of BrdUrd to the culture medium, metaphase chromosomes from only 3 of the 8 individuals studied showed increased levels of exchange. These results indicate that chlorpromazine can induce SCEs in vitro but that there is considerable variation in SCE response among individuals. Furthermore, our data emphasises the importance of using more than 1 or 2 donors when analysing SCE response in human chromosomes.     

In vivo and in vivo/in vitro kinetics of cyclophosphamide-induced sister-chromatid exchanges in mouse bone marrow and spleen cells

In several acute and chronic exposures to various chemicals in vivo and in vitro, the average sister-chromatid exchange (SCE) frequencies in human, mouse, rat, and rabbit lymphocytes generally decrease with time following treatment. The rate of this decline varies, but little data have been published pertaining to the comparative kinetics of SCEs both in vivo and in vivo/in vitro (exposure of animals to the test compound and culturing of cells) simultaneously in the same tissues. In this study, a single dose of cyclophosphamide (40 mg/kg) was injected for varying periods (6-48 h) and its effects, as assessed by the induction of SCEs, were analyzed under both in vivo and in vivo/in vitro conditions in mouse bone marrow and spleen cells. In vivo, the cyclophosphamide-induced SCEs increased with increasing time up to 12 h, stayed at approximately the same level until 24 h, and then decreased with increase in post-exposure time. However, the SCE levels remained significantly higher than controls at 48 h post-exposure time in both bone marrow and spleen cells. Under in vivo/in vitro conditions, the SCEs in bone marrow decreased with increase in post-exposure time until reaching control values by 48 h post exposure. However, in spleen cells, the decrease in SCE level was gradual, and by 48 h post-exposure time, the cells still had approximately 6 times higher SCEs than the control values. These results suggest that there are pharmacokinetic differences for cyclophosphamide in mouse bone marrow and spleen. Also, there is a differential SCE response to cyclophosphamide under in vivo and in vivo/in vitro conditions.     

抚仙湖有机污染物化学背景值及人淋巴细胞体外致突变性评定

用ＸＡＤ－２树脂提取抚仙湖７个采样点的有机物质水样提取物,用气相色谱／质谱（ＧＣ／ＭＳ）进行分析,在每种水样品中发现有上百种有机污染物,提取物用培养的人淋巴细胞系姐妹染色单体交换（ＳＣＥ）试验作致突变潜力检查,结果显示,仅入湖口水样在高浓度组（０．０６２５Ｌ／ｍＬ）诱发ＳＣＥ明显增加。     

Dependency of the yield of sister-chromatid exchanges induced by alkylating agents on fixation time. Possible involvement of secondary lesions in sister-chromatid exchange induction

Chinese hamster V79 cells were pulse-treated (for 60 min) with various mutagens three, two or one cell cycles before fixation (treatment variants A, B and C, respectively) and the frequencies of induced SCEs were analysed and compared. The degree of increase in frequency of SCEs with dose in the treatment variants depended on the mutagen used. For the methylating agents MNU, MNNG and DMPNU, high yields of SCEs were obtained in the treatment variants A and B, and there was no difference in the efficiency with which these agents induced SCEs in these treatment variants. In the treatment variant C, however, no SCEs were induced with mutagen doses yielding a linear increase in SCE frequency in treatment variants A and B. A slight increase in SCE frequency in treatment variant C was observed only when relatively high doses of MNU or MNNG were applied. Like the above agents, EMS, ENU and MMS induced more SCEs in treatment variants A and B than in C, but for these agents treatment variant B was most effective and SCEs were induced over the entire dose range, also in treatment variant C. As opposed to the methylating and ethylating agents, MMC induced SCEs with high efficiency when treatment occurred one or two generations prior to fixation. There was no difference in SCE frequency between these treatment variants. MMC was completely ineffective for the induction of SCEs when treatment occurred three generations before fixation. The unexpectedly low SCE frequencies induced by the methylating and ethylating agents when treatment occurred one generation before fixation were not due to the exposure of cells to BrdU prior to mutagen treatment. From the results obtained, it is concluded that DNA methylation and ethylation lesions give rise to SCEs only with very low probability during the replication cycle after the lesion's induction, and that subsequent lesions produced during or after replication of the methylated or ethylated template (secondary lesions) are of prime importance for SCE formation after alkylation. For MMC, however, primary lesions seem to be most important for SCE induction.     

Bimodal distribution of sensitivity to SCE induction by diepoxybutane in human lymphocytes. II. Relationship to baseline SCE frequency

Environmental and genetic factors have been implicated as important sources of individual variation in baseline sister-chromatid exchange (SCE) frequency in humans. The current study was designed to test whether the frequency of baseline SCEs in 58 normal blood donors is associated with previously observed variations in SCE frequencies induced by diepoxybutane (DEB). Because 12 subjects were current cigarette smokers and smoking is known to be an in vivo inducer of baseline SCE frequencies, we specifically tested whether higher baseline SCE frequencies in smokers would be associated with in vitro sensitivity to SCE induction by DEB. Analysis of variance showed that DEB-induced SCE frequencies were significantly associated with baseline SCE frequencies; those who were sensitive to SCE induction by DEB were more likely to have higher baseline SCE frequencies. This effect, however, was independent of in vivo induction of SCE by smoking. Chromosomal sensitivity to the induction of SCE by DEB explained approx. 15-20% of the variation in baseline SCE. This was similar in magnitude to the effect of cigarette smoking. Because increased sensitivity to DEB-induced SCEs is common in normal blood donors (approx. 24%) and is associated with an increase in baseline SCEs, it should be investigated as a source of bias and/or a potential marker of sensitivity to environmental mutagens in future cytogenetic studies.     

Diagnostic insonation of extra utero human placentas: No effect of lymphocytic sister chromatid exchange

Summary Freshly delivered human placentas were exposed to ultrasound for 30 min using a diagnostic linear array unit. Blood was then drawn and cultured in the presence of bromodeoxyuridine, and the frequencies of sister chromatid exchanges (SCE) in the lymphocytes determined. There was no statistically significant difference in SCE frequencies between control and exposed cells; the frequencies of SCEs per cell ranged from 4.50 to 6.02 for control and from 4.66 to 6.10 for exposed cells in five separate experiments. Positive control mitomycin C treated cells were significantly affected, with more than 50 SCEs per cell.     

Effects of temperature on chemically induced sister-chromatid exchange in human lymphocytes

Lymphocytes from healthy adults were studied for sister-chromatid exchanges (SCEs) when pulse-treated in G0 with mitomycin C (MMC), ethyl methanesulfonate (EMS), or 4-nitroquinoline N-oxide (4NQO) at various temperatures ranging from 0 degrees C to 41 degrees C and then cultured in medium containing 5-bromodeoxyuridine at 37 degrees C. The results showed that the frequencies of SCEs induced by MMC or EMS varied according to the treatment temperature. In MMC- or EMS-exposed cultures, the SCE frequency increased continuously with increasing treatment temperature; treatment at 37 degrees C resulted in a 3-4 times greater induction of SCEs than did that at room temperature (25 degrees C). On the other hand, SCE frequencies in cells exposed to 4NQO remained within normal deviation, showing no temperature-dependent changes. Baseline SCE frequencies remained almost constant within the temperature range tested. These data indicate that treatment temperature is a very critical factor in determining the sensitivity of cells to the chemical induction of SCEs.