Reduced frequencies of Mitomycin-C induced sister chromatid exchanges in AKR Mice

Summary The frequencies of base-line and Mitomycin-C (MMC) induced sister chromatid exchanges (SCE) were surveyed in four inbred strains of mice. In contrast to the C57B1/6J, CBA/J, and A/J strains where frequencies of SCE increased linearly with increasing dose of MMC, levels of SCE were significantly lower in AKR/J mice at high MMC concentrations. At a dose of 5 mg/kg MMC, chromosomal aberrations were more frequent in bone marrow cells of AKR/J mice than in C57B1/6J mice. These observations suggest an altered response to DNA damage in the AKR mouse strain.     

Dynamics of sister chromatid exchange after in vivo exposure to thiophosphamide

Thiophosphamide (T) was i.v. administered into New Zealand rabbit (4 mg/kg). The rate of SCE in blood lymphocytes was increased up to 21st day after T administration. There are two phases in dynamics curve of SCE--a rapid phase and a slow one. The rapid decrease of SCE's rate depends on cell mortality. The slow decrease of SCE rate is connected with reparation of chromosome damages and selection of cells in lymphopoietic tissue. It was demonstrated that the special class of cells with intermediate number of SCE exists in blood after T administration.     

Effect of mouse age and genotype on the frequency of sister chromatid exchange in bone marrow cells

No differences were found in both the baseline and mitomycin C induced levels of sister chromatid exchanges (SCE) between 101/H and C57BL/6J mice differing in chromosome mutability. An increase with the age of the spontaneous and mutagen induced SCE rates was similar in the strains compared, though instability of chromosomes was much higher in old 101/H than in C57BL/6J mice. Thus, no correlation was observed between chromosomal aberration and SCE levels in these strains. As 101/H mice were recently found to be DNA repair-deficient, possible connection of SCE and repair is discussed.     

Environmental monitoring for genotoxicity: in vivo sister chromatid exchange in the house mouse (Mus musculus)

Sister chromatid exchange (SCE) values were determined in bone marrow cells isolated from mouse (Mus musculus) femurs after injections of 5-bromo-2'-deoxyuridine (BrdU) and 5-fluorodeoxyuridine (FrdU). Male mice of C3H/J, C57BL/6J, and DBA/2 strains maintained in the laboratory gave mean SCE values of 3.42 +/- 0.07, 3.62 +/- 0.08, and 3.97 +/- 0.13, respectively. Males obtained from natural populations of southwestern Ontario had a higher mean SCE value (6.02 +/- 0.16), as did inbred males maintained in outdoor enclosures for at least 3 weeks (5.07 +/- 0.22). Wild mice housed in the laboratory for 9 months or longer had SCE values similar to laboratory bred mice (3.46 +/- 0.05). The SCE values in wild-caught mice were inversely proportional (r = -0.49) to the distance between the sites where these animals were collected and the nearest major industrial center. Based on these results, SCE analysis in mice is proposed as a possible first-line monitoring procedure for the detection of general changes in environmental genotoxicity.     

The measurement of sister chromatid exchanges induced in utero utilizing intraperitoneal infusion of BrdU: a novel technique

One of the inherent difficulties associated with the accurate detection of sister chromatid exchanges (SCE's) in vivo is the maintenance of a continuous supply of 5-bromo-2'-deoxyuridine (BrdU) for differential staining of sister chromatids. We have developed a simple, reproducible intraperitoneal technique which has the advantages of continuous BrdU infusion and is less stressful than presently available methods. Additionally, a gentle trypsinization methodology was developed to obtain relatively large numbers of metaphases from fetal tissues. Using these techniques, we examined the induction of SCE by 7,12-dimethylbenz(a)anthracene (DMBA) when injected into pregnant CD rats on days 11, 13, 15, and 16 of gestation. The levels of SCE induced were slightly higher in fetal than maternal tissue, although not significantly.     

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.     

Effects of temperature on sister chromatid exchanges

Summary The influence of temperature on sister chromatid exchanges was investigated, and the results are discussed in connection with factors possibly involved in temperature-induced SCE-formation.Whereas the SCE frequency increased with increasing growth temperature in a cell line of Xenopus laevis (EAX), which permits the examination of great temperature differences, a Chinese hamster cell line (V-79) revealed a U-shaped temperature-response curve. In addition, it was found that cold treatment at 4°C caused an induction of SCEs in the V-79 cell line.Different BrdU concentrations had no effect on the temperature-induced SCE frequencies and mitomycin C led to an induction of SCEs parallel to the base-line values at different temperatures.     

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     

Sister chromatid exchanges and lack of isolabeling in Chinese hamster chromosomes

Chinese hamster CHO and Don cells were synchronized in mitosis with or without a Colcemid treatment, labeled with ³H-TdR through an entire cell cycle, and were synchronized again each time they entered mitosis. Over four cycles without label, only heterolabeling patterns were observed, and the frequency of sister chromatid exchanges (SCE's) per chromosome per cycle (0.23 or 0.02/μm) was constant. After the labeling cycle, during which the total SCE frequency was also 0.23, a disproportionately high SCE frequency at the centromeres, about 0.15, decreased dramatically to about 0.03, while the SCE frequency in the arms increased from about 0.08 to 0.20. In accord with reports in the literature, the constant SCE frequency of 0.23 appeared to be the saturation frequency resulting from the ³H disintegrations. It was postulated that during the labeling cycle, the limited number of SCE's occurred preferentially at the centromere because ³H disintegrations occurring in the daughter strand of DNA produced alterations which were preferentially repaired by a recombinational SCE mechanism. Implicit in this hypothesis is the assumption that SCE's result from the transmutation or recoil effect of the ³H disintegration and that there is a unique association between the kinetochore and the daughter strand of DNA.     

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

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 mechanisms(s) of SCE formation in FR3T3 cells are largely temperature independent (or efficiently regulated) in the range between 33 and 40.5 degrees C. (b) The concentration limits for BrdU incorporation are 5 to 100 microM; baseline frequency is about 11 SCE/metaphase (constant up to 20 microM BrdU) and increases only moderately at higher BrdU concentrations. (c) Toxic levels of BrdU (150 microM) cause a decrease of SCE rates below that found at 100 microM, 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.     

The co-induction of sister chromatid exchanges and virus synthesis in mammalian cells

The induction of virus synthesis and sister chromatid exchange (SCE) formation was investigated in several mammalian cell lines. Ultraviolet light co-induced the production of virus and SCEs in Simian virus 40 (SV40) transformed hamster cells. Post-irradiation treatment with caffeine enhanced virus induction, though it caused a smaller, less consistent elevation of SCE formation. Co-induction of oncovirus synthesis and SCEs was also observed in three murine cell lines exposed to increasing concentrations of 5-bromodeoxyuridine. These and previous data demonstrate a correlation between the induction of virus synthesis and SCE formation in rodent cells exposed to several agents, although inter-agent variation in the correlation may reflect differences between the two processes.     

Differential giemsa staining of sister chromatids and the study of sister chromatid exchanges without autoradiography

Chinese hamster ovary cells grown for two rounds of DNA replication in the presence of BrdUrd contain sister chromatids that fluoresce differentially when stained with Hoechst 33258. If such fluorescent treatments are followed by incubation in 2 X SSC or water at 62° C and staining in 3% Giemsa, the chromosomes now contain one dark (unifilarly substituted) chromatid and one light (bifilarly substituted) chromatid, i.e. are harlequinized. These preparations do not fade and can be studied without resorting to fluorescence microscopy. Sister chromatid exchanges (SCE's) are seen with great clarity and resolution; and all the chromosomes in a cell can be scored, which is contrary to the usual experience with autoradiography. It was found that a) the yield of SCE's is dependent upon the concentration of BrdUrd in which the cells are grown and that the maximum number of SCE's that can occur spontaneously is 0.15 per chromosome per division cycle, b) the yield of SCE's doubles if the cells are exposed to visible light that can cause the photolysis of BrdUrd-containing DNA, and c) chromosomes that appear isolabelled in autoradiographic preparations come from observable multiple exchanges and are not the result of the segregation of DNA from a binemic chromosome. Furthermore, the staining patterns obtained in endoreduplicated cells clearly confirm that the polynucleotide strands of the DNA segregate into sister chromatids as though the newly synthesized strands were laid on the outside of the replicating double helix.     

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.     

Frequency of sister chromatid exchanges in severe protein calorie malnutrition.

Nine children with severe protein calorie malnutrition were studied regarding the frequency of sister chromatid exchanges (SCE's) in peripheral blood lymphocytes. The results showed that there was no significant difference between the number of SCE's in the malnourished children as compared to an adequate control group. An interesting finding was that the proportion of 3rd or subsequent division metaphases found in the malnourished children, was higher and significantly different from that seen in the control group.     

Induction of sister chromatid exchanges and chromosome aberrations in cho cells arrested in the cell cycle by arginine deprivation

Summary Sister chromatid exchanges (SCE) and chromosome aberrations were induced in nondividing CHO cells that had been arrested in their cell cycle by deprivation of the essential amino acid arginine. Cells arrested in arginine-deficient medium (ADM) were treated with one of the mutagenic agents N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) or mitomycin C (MMC) and refed with complete medium; the recovering cell population was sampled at various intervals thereafter and mitotic cells analyzed for the presence of chromosome aberrations and SCE. Both chemicals were observed to cause delays in the cell cycle of recovering cells and to induce, chromosome aberrations and SCE at low doses. We have described the variation in the incidence of chromosome aberrations and SCE with respect to sampling time and the number of cell cycles traversed. When ADM-arrested CHO cells were treated with three mutagens at various intervals either before or after release from ADM, it was observed that: (a) UV light induced the greatest number of SCE when applied to cells undergoing DNA synthesis, and SCE yeilds induced by this agent could be reduced by postirradiation incubation in ADM; (b) MNNG induced fewer SCE when applied to cells undergoing DNA synthesis, and SCE yields induced by this agent could not be reduced by posttreatment incubation in ADM for 24 hr. (c) MMC induced the same level irrespective of the time of exposure, and SCE yields induced by this agent could not be reduced by posttreatment incubation in ADM for 24 hr. This work was supported by grants from the British Columbia Foundation for Non-Animal Research (to W. D. M.), and the National Cancer Institute of Canada and the National Research Council of Canada (to H. F. S.). Professor H. F. Stich is a Research Associate of the NCI.     

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.     

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.     

The interaction of Hoechst 33258 and BrdU substituted DNA in the formation of sister chromatid exchanges

Sister chromatid exchanges (SCEs) are induced in cultured Chinese hamster cells by treatment with 5-bromodeoxyuridine (BrdU) or with Hoechst 33258 (H33258) plus BrdU. The SCE frequencies depend upon the number of H33258 molecules available per cell (or per base pair) and the number of brdU molecules available per cell, and not solely upon molarity. In addition, H 33258 and BrdU act synergistically to induce SCEs. At low BrdU concentrations H33258 induces very few SCEs. At high BrdU concentrations and similar concentrations of H33258, however, SCE frequencies are significantly increased. SCE frequencies decrease with time in successively harvested cells because of the depletion of H33258 from the medium due to DNA binding.     

Induction of endoreduplication by hydrazine in Chinese hamster V 79 cells and reduced incidence of sister chromatid exchanges in endoreduplicated mitoses

Hydrazine in high concentrations very effectively induces endoreduplication in Chinese hamster V 79 cells. The addition of 5-bromodeoxyuridine (BrdU) for the duration of one cell cycle prior to the induction of endoreduplication produces diplochromosomes with sister chromatid differentiation (SCD) after differential chromatid staining. The fact that diplochromosomes with complete SCD are obtained shows that endoreduplication was induced in cells that were in G2-phase. The analysis of sister chromatid exchanges (SCEs) showed that hydrazine treatment rarely led to increased SCE frequencies in mitoses after endoreduplication, but that it caused a strong SCE induction in diploid second division metaphases in the same culture. Neither catalase nor cysteine had an effect on the induction of endoreduplication or the incidence of SCEs. Treatment of the cells with mitomycin C prior to addition of BrdU led to increased SCE frequencies. Compared with the normal mitoses from the same preparation, the mitoses after endoreduplication showed a significantly reduced induction of SCEs. In contrast to these findings, SCE induction was not reduced in the common tetraploid V 79 cells after colcemid-induced polyploidization.     

Analysis of bromodeoxyuridine-induced single and twin sister chromatid exchanges in tetraploid Chinese hamster ovary cells

Culture of cells in high exogenous levels (>10^–4 M) of bromodeoxyuridine (BrdUrd) or thymidine will increase the baseline sister chromatid exchange (SCE) frequency. The effect is thought to be related to the balance of the DNA precursors thymidine and deoxycytidine. Exogenous addition of deoxycytidine will reverse this effect. Single and twin SCEs were analysed in Colcemid-induced tetraploid Chinese hamster ovary cells exposed to different concentrations of BrdUrd to determine at what stage SCEs are induced by high levels of BrdUrd. In cells exposed to low concentrations of BrdUrd (10^–5 M), equal numbers of SCEs were induced in each of the two cell cycles. With increasing concentrations of BrdUrd (10^–4 to 2×10^–4 M), SCE frequency increased in both cell cycles, but far more SCEs were induced in the second cell cycle. Deoxycytidine (2×10^–4 M) reduced the frequency of SCEs primarily by reducing the frequency of SCEs induced in the second cell cycle. Treatment with 3-aminobenzamide (3AB), a potent inhibitor of poly(ADP-ribose) polymerase, produced effects similar to exposure to high levels of BrdUrd including inducing SCEs in the second replication cycle. This suggests a similar mechanism of action. Deoxycytidine had no effect on 3AB-induced SCEs, however, and there was no interaction between 3AB and high exogenous levels of BrdUrd in SCE induction. Thus these two agents probably act through different mechanisms.