Sister chromatid exchanges induced by cyclophosphamide in V79 cells cultured in diffusion chambers in mice

Chinese hamster cells V79 were cultured in diffusion chambers (DC) and implanted into mice. An exponential growth was observed from the 2nd to 4th day after implantation. The maximum growth was reached on the 6th day. After that, cell growth and viable cell counts decreased. Three days after implantation of DC with V79 cells, the hosts received 6 hourly injections of 0.2 ml of 5-bromodeoxyuridine (BUdR) solution at concentrations of 0.125 to 1.0 x 10(-2) M. DC were removed for chromosome and sister-chromatid exchanges (SCE) analyses 24 h after the first BUdR injection. The frequency of metaphases with differentially stained chromatids, with aberrations, and the number of SCE per cell increased with BUdR dose. The frequency of metaphases with differentially stained chromatids was also positively correlated with the duration of BUdR exposure or the number of hourly injections of BUdR-solution. The effects of cyclophosphamide (CY) in V79 cells in DC in mice were studied. Injections of CY at 2.5, 5, 10 and 15 microgram per gram of body weight to the hosts caused an increase in the number of SCE per cell in a linear manner. The results from this study indicate that V79 cells cultured in DC in mice may provide a potential test system for mutagenicity.     

Induction of sister-chromatid exchanges in Chinese hamster ovary cells by thiol and hydrazine compoudns

Cysteine, cysteamine and glutathione all induce sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells when applied to cell cultures at concentrations between 10(-4) and 10(-2) M. Acute exposure of cells th thiol compound for a period of 2--3 h resulted in a unique dose--response relationship in each instance. This consisted of two peak SCE frequencies, one at either extreme of the concentration range. Each peak corresponded to a 2--3-fold increase over the spontaneous level. A chronic exposure of 24 h, in contrast, resulted in a dose--response relationship consisting of a single peak SCE frequency (representing a 4--5-fold increase over the spontaneous level) at a concentration of approx. 4 x 10(-4) M. The effect of Cu2+ ions included in the medium at a concentration of 10(-5) M was to increase the toxicity and, at some concentrations, the SCE levels occurring after either acute or chronic exposure to thiols. Hydrazine and its derivatives, dimethylhydrazine and isonicotinic acid hydrazide (isoniazid), as well as hydrogen peroxide, also induce SCEs in CHO cells. A 2--3-fold increase over the spontaneous level was observed, depending upon the particular treatment protocol applied. SCE yields after 3 h treatment with dimethylhydrazine and isoniazid were increased if Mn2+, but not Cu2+, was included in the tissue culture medium at a concentration of 10(-5) M. SCE yields after a 24-h treatment with dimethylhydrazine in which Mn2+ was present in, and absent from, the medium were similar. Catalase was observed to reduce the SCE levels resulting from treatment with hydrogen peroxide, dimethylhydrazine and isoniazid. The effect of catalase upon SCEs induced by dimethylhydrazine and isoniazid in the presence of Mn2+ was more evident than when Mn2+ was not included in the culture medium. The significance of these results with respect to the possible active chemical species produced and the mutagenic/carcinogenic risk associated with thiol and hydraizine compounds is discussed.     

Demonstration of spontaneous sister chromatid exchanges in vivo

The frequency of sister chromatid exchanges (SCEs) was examined as a function of bromodeoxyuridine (BUdR) concentration in vivo. Oneyear-old Wistar rats were continuously infused with BUdR through the tail vein for 24 h, sacrificed, and mitotic preparations prepared from femur bone marrow. It was observed that from the minimum concentration of BUdR which permitted accurate scoring (1.9 μg/g wt/h) to a BUdR concentration of 7 μg/g wt/h, SCE frequency remained constant. Above 7 μg BUdR/g wt/h SCE frequency increased, saturating at higher BUdR concentrations. The stability of SCE frequency at low BUdR concentrations is interpreted to indicate the existence of spontaneous SCEs in vivo.     

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.     

Relationship between cytotoxicity and induction of sister-chromatid exchanges in mouse foetal cells exposed to several doses of carcinogenic and non-carcinogenic chemicals.

The increase in sister-chromatid exchanges induced by 5 chemicals, with different DNA damaging and carcinogenic activities, was studied in short-term foetal-mouse cultures. A significant increase in SCE was induced by N-methyl-N'-nitro-N-nitrosoguanidine, N-diazoacetylglycine-amide, azaserine and methotrexate. k-Strophantin, on the contrary, was totally inactive. On a molar basis, MNNG was the most active chemical followed by MTX, AZS and DGA, in that order. At equitoxic concentrations (D37), the order of SCE-inducing abilities was MNNG, DGA, AZS and MTX. Compared with previous data, at equitoxic concentrations, the most DNA-damaging agents were also the most effective in inducing SCE. The SCE increase seems to correlate not with unspecific cytotoxicity but more with DNA damage or other damage at the genome level. MTX, a non-mutagen, which induced SCE only at toxic levels, could be considered a false positive because this positivity may reflect an enhancement of incorporation of 5-BrdUrd into DNA. The positive results obtained with AZS suggest a sufficient sensitivity of the method for detecting relatively weak carcinogens.     

Genotoxic effects of thiram evaluated by sister-chromatid exchanges in human lymphocytes

The purpose of this investigation was to study the genotoxic potential of thiram (CAS No. 137-26-8) using an in vitro sister-chromatid exchange (SCE) assay with human lymphocytes. The results indicate that thiram and its metabolites increase the SCE frequencies 2-fold over those observed in the negative controls. The standard inducers cyclophosphamide and ethyl methanesulfonate increased SCE frequencies 10- and 4-fold, respectively, over untreated levels.     

Analysis of sister chromatid exchanges in lymphocytes cultured from 71 healthy men

Sister chromatid exchanges (SCE) were analyzed in peripheral blood lymphocytes from a select group of 71 healthy men, 56 nonsmokers and 15 cigarette smokers. In addition to estimating baseline SCE, data were examined to seek relationships of SCE frequencies to age and smoking. The baseline value of 7.53 SCE per cell from the 56 nonsmokers was within the range (5.60 to 9.10 SCE/cell) reported for other human populations. No relationship was found between the mean SCE frequency per cell and age. However, a significant increase in the SCE mean value was observed in smokers as compared to nonsmokers. The results of this study are compared with those of other reports on SCE effects of age and smoking.Abbreviations BUdR 5-bromo,2-deoxyuridine - SCE sister chromatid exchange     

Effect of incubation temperature on the frequency of sister chromatid exchanges in Bloom's syndrome lymphocytes

Summary The effect of incubation temperature on the frequency of sister chromatid exchange (SCE) has been studied in blood cultures from three Bloom's syndrome (BS) patients, three controls, and three BS heterozygotes. All cell types show slight increases of SCE at 39°C while at 35°C and 32°C, SCE is reduced considerably in BS and slightly increased in normal cells. Prolonging lymphocyte culture to 140 h and adding BUdR for the last two S periods causes a similar decrease in the percentage of SCE in normal and BS cells but, while the latter show a further reduction if they are incubated at 32°C during BUdR labelling, the normal cells show an increase. Therefore, BS and control lymphocytes respond similarly to changes in incubation time and differently to changes in incubation temperature. The possibility that the discrepant behaviour of the BS and control cultures may be due to different growth kinetics of their B and T lymphocytes has been discussed but considered unlikely. Since low temperature lengthens the cell cycle, it has been suggested that our findings and those published by others on co-cultivation experiments (except those of Tice et al. 1978) can be explained by assuming that slow growth reduces SCE in BS cells. This, and unpublished observations (Giannelli et al. 1981), suggest that some imbalance in the factors responsible for DNA replication may exist in BS and possibly account for the high level of SCE.     

Sister-chromatid exchange studies on direct- and indirect-acting clastogens in mouse primary cell cultures

An in vitro sister-chromatid exchange (SCE) assay using mouse primary bone marrow and spleen cells was conducted with both direct- and indirect-acting genotoxic agents. 2,4,7-Trinitrofluorenone, a direct-acting genotoxic agent, induced a significant dose-related increase in SCEs. In both bone marrow and spleen cells, 2.0 micrograms/ml caused an approx. 3-fold increase in SCE level over control values. Cyclophosphamide, an indirect-acting genotoxicant which requires metabolic activation for its clastogenicity, induced a significant increase in SCEs in the presence of S9 from liver of rats pretreated with Aroclor-1254. A dose of 2 micrograms/ml resulted in a 2-fold increase in bone marrow and a greater than 5-fold increase in spleen cells. Benzo[a]pyrene, another indirect-acting genotoxicant, also induced significant dose-related SCE responses in both cell types. It seems that primary bone marrow and spleen cell culture systems can detect both direct- and indirect-acting genotoxicants and may be useful for routine and/or comparative cytogenetic studies.     

Sister chromatid exchanges in human leukocyte chromosomes: Spontaneous and induced frequencies in early- and late-proliferating cells in vitro

Summary Human leukocyte cultures were pulse-treated with the trifunctional alkylating mutagen trenimon in a final concentration of 10^-7 M for 15–20 h after culture start, i.e., in the G₁ phase of the cell cycle. At 24 h after culture start bromodeoxyuridine (BUdR) was added to the trenimon-treated cultures and to several untreated cultures running in parallel. The series treated with BUdR only and the series treated with BUdR+trenimon were each used to prepare two cultures at different culture times. Mitoses were collected during consecutive intervals of 12 h from 30 h up to 102 h after culture initiation by colcemid. For all preparation times (42 h, 54 h, 66 h, 78 h, 90 h, and 102 h) the frequencies of first, second, and third and further mitoses were determined in the BUdR- and in the BUdR+trenimon-treated series. In the trenimon-treated series a clear cell cycle delay was detected as compared with the normal distribution of different types of mitoses found in series treated with BUdR only. Spontaneous and trenimon-induced sister chromatid exchange (SCE) frequencies were determined in second mitoses occurring at 66 h, 78 h, 90 h, and 102 h after culture start. For all these preparation times about six SCE per metaphase were consistently found in BUdR-treated, and about 19 SCE per metaphase in BUdR+trenimon-treated series, indicating a homogeneous sensitivity of early- and late-proliferating cells with respect to the induction of SCE.This paper is dedicated to Prof. B. Erich Wolf on the occasion of his 70th birthday     

Sister chromatid exchange in methotrexate resistant and sensitive C3H10T1/2 mouse cells

Sister chromatid exchange (SCE) induction by methotrexate (MTX) was analyzed in C3H10T1/2 clone 8 mouse cells and in two MTX-resistant subclones with numerous double minute chromosomes (DM) present in the majority of cells. Significantly higher SCE levels were found, as expected, in sensitive cells after treatments with 10^-2 or 10^-5M MTX but not in resistant cells permanently growing in the presence of a high concentration of MTX (2×10^-3M) and characterized by a markedly lower cell cycle replication index (R.I.), i.e. in conditions that are known to otherwise favour SCE induction. These observations suggest, for the MTX-resistant cells under study, the existence of conditions limiting SCE formation.     

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.     

A simple technique for in vivo observation of SCE in mouse ascites tumor and spermatogonial cells

A simplified technique to observe sister chromatid exchange (SCE) in ascites tumor cells and spermatogonial cells of the mouse was described. The technique consisted of a single intraperitoneal injection of 5-bromo-2′-deoxyuridine (BUdR) absorbed on activated charcoal that assured continuous release of BUdR throughout two rounds of the replication period. The minimum dose of BUdR to detect SCE was found to be 5 mg/body, the SCE frequency being 4.50 – 4.90/ cell in tumor cells and 1.30 – 1.88/cell in spermatogonia, respectively.     

Spontaneous level of sister chromosome exchanges and their distribution in human cells]

Blood of practically healthy donors of both sexes (27 females and 23 males) was cultured under the standard conditions during 96 hours. Bromodeoxyuridine (BUdR) was added at the final concentration of 10 mkg/ml 28 hours before harvesting. The slides were stained with acridine orange and Giemsa for differential staining of chromatids. In each culture sister chromatid exchanges (SCE) were analysed in 50 cells, and the part of cells undergoing the first, second and third mitoses at the time of harvesting, was calculated. According to the mean number of SCE per cell, the distribution of individuals was consistent with the normal law, the mean being 6.525 and standard deviation--0.956. A significant heterogeneity in the speed of cell cycle of cultures was observed. The coefficient of variation for the part of cells undergoing the first mitosis was 50%, for the cells in the second mitosis--15%, and for the cells in the third mitosis--154%. Correlation analysis showed a positive dependence of the mean level of SCF upon the age of a donor and upon the part of cells in the second mitosis in this individual. No reliable correlation of the SCE level with the donor's sex was observed. The distribution of cells, obtained from the culture of one individual, was best approximated by beta-distribution, and the distribution of cells obtained from the cultures of different individuals--by gamma-distribution. In both there was obtained a satisfactory approximation by Pearson's distribution of the 1 type, and significant deviations were found from the normal, Poison's and the negative binomial distribution. The conditions were found of similarity of empirical distribution of SCE in cells to the normal one. For that, it is not the value of SCE for a separate cell that should be used as a unit of measurement, but the mean from the values of frequencies for 5-10 cells. Hence, it was shown that for the evaluation of the mean frequency of SCE with the precision of 1 exchange in separate individuals it is necessary to analyse 40 cells, and to observe the 15% increase of spontaneous SCE level under the action of deleterious factors--8 individuals are enough to analyse.     

Decrease in the spontaneous frequency level of sister chromatid exchanges in cell division in culture

The spontaneous level of sister chromatide exchanges (SCE) registered in human lymphocytes is shown to depend on the moment of BUdR introduction and the time of fixation. In early periods of BUdR introduction and fixation the general spontaneous level of SCE may be observed and in later periods only that part of SCE may be registered which is caused by internal conditions. The difference between the first and second results makes the part of SCE conditioned by the environmental effects.     

Sister-chromatid exchanges in lymphocytes of workers at a phosphate fertilizer factory

The frequencies of sister-chromatid exchange (SCE) in peripheral blood lymphocytes of 40 workers at a phosphate fertilizer factory in North China were studied. HF and SiF₄ are main air pollutants in the factory, there is also some dust containing fluoride, phosphate fog, NH₃ and SO₂. It was shown that the chemicals caused an increase in SCE, and also induced cell mitotic delays. The mean SCEs/cell of the workers and the non-exposed controls were 7.47 ± 0.31 and 4.94 ± 0.14 (p < 0.01) respectively. SCEs/cell in 75% of 40 workers were higher than 6 while 40 controls all had values lower than 6. SCE frequencies of the workers increased with length of the chemical exposure period up to 10 years. Smoking enhanced the SCE frequencies induced by the chemicals.     

Regulation of rat myosin light-chain synthesis in heterokaryons between 5-bromodeoxyuridine-blocked rat myoblasts and differentiated chick myocytes

Terminal cell differentiation in a variety of model systems is inhibited by the thymidine analogue 5-bromodeoxyuridine (BUdR). We investigated the mode of action of BUdR by forming heterokaryons between undifferentiated BUdR-blocked rat myoblasts and differentiated chick skeletal myocytes. We analyzed newly synthesized proteins on two- dimensional polyacrylamide gels. The induction of rat skeletal myosin light-chain synthesis was reduced fivefold, as compared with controls, when chick myocytes were fused to BUdR-blocked rat myoblasts. This indicates that plasma membrane effects cannot be the proximate cause for the inhibition of myogenesis by BUdR, since BUdR is able to block the effect of chick inducing factors even when a differentiated chick myocyte is in direct cytoplasmic continuity with the BUdR-blocked rat nucleus. The observation that chick cells required an 80% substitution of BUdR for thymidine to block myogenesis, whereas L6 rat myoblasts required only a 20% substitution led to a hypothesis involving a DNA- mediated action of BUdR. This model yielded three testable predictions: (a) putative chick inducing molecules should be present in limiting quantities, (b) exploiting gene-dosage effects to increase the quantity of putative chick inducing factors might overcome the inhibition produced in the rat myoblasts by a 35% BUdR for thymidine substitution, and (c) these gene-dosage effects should be abolished by increasing the level of BUdR substitution in the rat myoblast to 60-80%. All three of these predictions have been verified, providing strong indirect evidence that the inhibition of myogenesis produced by BUdR is a direct result of its incorporation into cellular DNA.     

Investigations into sister chromatid exchange in patients under cytostatic therapy

Summary In vivo sister chromatid exchange (SCE) determinations were carried out in patients with Psoriasis vulgaris under methotrexate therapy and patients with histologically verified bronchial carcinoma under cyclophosphamide, methotrexate or a combined chemotherapy. A pronounced increase in SCE rate was only found after cyclophosphamide treatment.Abbreviation Cyclo cyclophosphamide - MTX methotrexate - FU fluorouracil - Pred prednisolone - VB vinblastine - VC vincristine - BM bleomycin     

Effects of mitomycin C on sister chromatid exchange in normal and Bloom's syndrome cells

Bloom's syndrome lymphocytes, which are characterized by a high incidence of sister chromatid exchanges (SCE: 80.6 per cell), were treated with mitomycin C (MMC) and the effect of the chemical on SCE frequency compared with that in normal cells. Raising the concentration of MMC from 1 X 10(-9) to 1 X 10(-7) g/ml led to about 10-fold increase (61.7 SCE per cell) in the SCE frequency over the base line in normal lymphocytes (6.4 SCE per cell), though chromosome aberrations remained at a relatively low frequency. MMC caused about a two-fold rise in SCE in cells of Bloom's syndrome (128.8 SCE at 10(-9) g/ml; 139.3 SCE at 10(-8) g/ml). The frequency of chromosome aberrations in Bloom's syndrome cells at concentrations of MMC of 1 X 10(-9) and 1 X 10(-8) g/ml was 0.350 and 0.825 per cell, respectively, and low when compared to the increased number of SCE. The increased frequency of SCE in normal and Bloom's syndrome cells is in contrast to the reported findings with cells from Fanconi's anemia and xeroderma pigmentosum. The distribution of SCE in MMC-treated normal cell correlates with that of spontaneous SCE in cells of Bloom's syndrome.     

Cytogenetic damage induced in human lymphocytes by sodium bisulfite.

The frequencies of chromosomal aberrations (CA), sister-chromatid exchanges (SCE), and micronuclei (MN) in human blood lymphocytes exposed to sodium bisulfite (sulfur dioxide) at various concentrations ranging from 5 x 10(-5) M to 2 x 10(-3) M in vitro were studied. It was shown that sodium bisulfite (NaHSO3 and Na2SO3, 1:3 M/M) caused an increase in SCE and MN in human blood lymphocytes in a dose-dependent manner, and also induced mitotic delays and decreased mitotic index. For CA, our results indicated that sodium bisulfite induced an increase of chromatid-type aberrations in lymphocytes from three of four donors in a dose-dependent manner. The chemical at low concentrations induced chromatid-type aberrations, but not chromosome-type aberrations; high concentrations induced both chromatid- and chromosome-type aberrations. No cytogenetic damage in human lymphocytes was induced by sodium sulfate. The results have confirmed that sulfur dioxide is a clastogenic and genotoxic agent.