Sister chromatid differentiation and exchanges in adult mudminnows (Umbra limi) after in vivo exposure to 5-bromodeoxyuridine

An in vivo system for the detection of sister chromatid exchange (SCE) in the central mudminnow, Umbra limi, is presented. Sister chromatid differential (SCD) and SCE were demonstrated by fluorescent and Giemsa procedures 5 to 6 days after the fish were injected with 500 g/g of BrdU. The exchange rate was found to be 2.64 SCEs metaphase in the intestines and 2.42 SCEs/metaphase in the gills. SCE analysis in U. limi should be a useful tool for measuring the mutagenicity of water-borne chemicals.     

SCE levels in Bloom-syndrome cells at very low bromodeoxyuridine (BrdU) concentrations: monoclonal anti-BrdU antibody

A stable staining procedure of sister-chromatid differentiation (SCD) using a monoclonal antibromodeoxyuridine (BrdU) antibody was newly established by combining it with the immunoperoxidase reaction (3,3'-diaminobenzidine, DAB reaction). This procedure permitted detection of SCD and SCE at very low BrdU concentrations. SCD was not usually observed below 2.0 micrograms/ml BrdU with flame-dried chromosome slides. When chromosome slides were prepared by air-drying over 37 degrees C warm water, SCD was detected at 10.0, 5.0, 1.0, 0.5, 0.3 and 0.2 micrograms/ml BrdU with FPG and even at 0.1 microgram/ml BrdU with the antibody technique. SCE levels were evaluated using the antibody technique and endomitotic analysis with FPG at low BrdU concentrations (1.0, 0.5, 0.3, 0.2 microgram/ml) in two BS B-lymphoblastoid cell lines (LCLs). Even though the BS SCE level was approximately 70 per cell at 10 micrograms/ml, the value decreased to the level of 20-30 SCE per cell at 0.1 microgram/ml with the antibody technique. In BrdU-labelled BS endomitoses, single SCEs highly decreased with BrdU concentrations (130-140 level at 10 micrograms/ml: 38-60 level at 0.2 microgram/ml), when compared to the rare twin SCE values (3-6 SCE level) at all BrdU concentrations. These findings conclusively indicate that the spontaneous baseline SCE in BS B-lymphoblastoid cells is low and most BS SCEs are caused by BrdU.     

Evidence for chromosomal replicons as units of sister chromatid exchanges

Chromosomal replicons have been described as the cytological counterpart of DNA replicon clusters and have previously been studied in vitro using premature chromosome condensation-sister chromatid differentiation (PCC-SCD) techniques. Chromosomal replicons are visualized as small SCD segments in S-phase cells, and measurement of these segments can provide estimates of relative chromosomal replicon size corresponding to DNA replicon clusters functioning coordinately in S-phase. Current hypotheses of sister chromatid exchange (SCE) formation postulate that sites of SCE induction are associated with active replicons or replicon clusters. We have applied the PCC-SCD technique to in vivo studies of mouse bone marrow cells that have been treated with cyclophosphamide (CP) for two cell cycles. We have been able to visualize chromosomal replicons, as well as SCEs which have been induced in vivo by CP treatment, simultaneously in the same cells. Chromosomal replicons visualized as small SCD segments were measured in PCC cells classified at early or late S-phase based on SCD segment size prevalence. Early S-phase (E/S) PCC cells contained 90% of the SCD segments measured clustered in a segment size range of 0.1 to 0.8 m with a peak value around 0.3 to 0.6 m regardless of CP treatment. As the cells progressed through S-phase, late S-phase (L/S) PCC cells were characterized by the appearance of larger SCD segments and even whole SCD chromosomes in addition to small SCD segments. A concentration of units around 0.4 to 1.0 m was found for L/S SCD segment size distributions regardless of CP treatment with an apparent bimodal profile. Our in vivo data support the existence of a subunit organization of chromosomal replication with a basic functional unit being 0.3 to 0.6 m in size. In addition, we have found that this chromosomal unit of replication or chromosomal replicon does not seem to be functionally perturbed by the mutagen CP. We also found that small SCD segments of 0.4 to 0.7 m in length were involved in the formation of an SCE, suggesting that both spontaneous and CP-induced SCEs occur between chromosomal replicons. These findings provide direct cytogenetic evidence to support a replicon cluster/chromosomal replicon model for SCE formation.     

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.     

Differential fluorescence of sister chromatids in chicken embryos exposed to 5-bromodeoxyuridine

Differential fluorescence of sister chromatids (SCD) and sister chromatid exchanges (SCE) were visualized in chromosomes obtained directly from growing chicken embryos. SCD was obtained by exposing 3-day embryos to BrdU (12.5-50 mug) in ovo for 26 hours and staining air dried chromosome preparations with 33258 Hoechst. Bright, stable fluorescence and continued SCD were achieved if slides were mounted in McIlvaine's pH 4.4 buffer. Embryo growth, mitotic activity and gross chromosome morphology were not adversely altered by the BrdU treatments. The SCE rate was estimated to be 0.07 SCEs per macrochromosome and 0.75 SCEs per metaphase for two cell cycles.     

5-bromodeoxyuridine tablets with improved depot effect for analysis in vivo of sister-chromatid exchanges in bone-marrow and spermatogonial cells

An improved 5-bromodeoxyuridine (BrdU) tablet technique for observation in vivo of SCE in mouse bone-marrow and spermatogonial cells is described.BrdU tablets were coated with agar as protecting barrier before subcutaneous implantation into mice. In comparison with the original tablets, the agar-coated tablets provided a slower and more uniform delivery of BrdU to the animals. This was corroborated (1) by recovering the undissolved portion of tablets at 1–2-h intervals, and (2) by quantitative determination of the BrdU levels in blood with the help of an analytical HPLC technique.The time required for complete dissolution of the coated tablets was considerably longer than that for the original tablets. This means that the dose of BrdU required for observation of SCE in mouse bone-marrow cells can be reduced accordingly. By using these modified tablets, therefore, undesired effects of high doses of BrdU on mutation (base-line SCE frequency) as well as on cellular replication and proliferation can be diminished.Moreover, the improved depot effect of the modified tablets facilitates the differential labeling of sister chromatids in mouse spermatogonia, a tissue containing cells with a relatively long DNA synthesis period.     

Comparison of urethane-induced sister-chromatid exchanges in various murine strains, and the effect of enzyme inducers

The induction of sister-chromatid exchanges (SCEs) by urethane, 150 and 300 mg/kg administered i.p., was examined in bone-marrow cells of AKR, BALB/c, C3Hf, C57BL/6J and DBA/2 male mice. In all strains, the base-line level of SCE/cell was similar, ranging from 4.3 to 8.7, and the response increased with the dose of urethane. DBA/2 mice were the most susceptible to urethane at both dose levels, with 30.6 SCE/cell after treatment with 300 mg/kg, whereas the response of the other strains was from 17.4 to 21.5 SCE/cell at the same urethane dose. Pretreatment of C57BL/6J and DBA/2 mice with phenobarbital decreased the SCE frequencies induced by urethane, 300 mg/kg, to 70%, whereas a prior administration of beta-naphthoflavone reduced SCE levels in C57BL/6J but not in DBA/2 mice.     

Some factors affecting sister-chromatid differentiation (SCD) and sister-chromatid exchange (SCE) in Hordeum vulgare.

A study of some factors affecting sister-chromatid differentiation (SCD) and sister-chromatid exchanges (SCE) in Hordeum vulgare is reported. After we studied the influence of 5-fluorodeoxyuridine (FdU) and growth temperature on SCE in barley cells, and the effect of FdU, growth temperature, the growth time of plant cells in 5-bromo-2-deoxyuridine (BrdU) solution on SCD, we found an experimental condition under which the frequency of SCE is lower, but the percentage of SCD is higher. Our data show that ascorbic acid, mitomycin C, adriamycin, and maleic hydrazide induce SCEs in cells of Hordeum vulgare by means of free radicals. This can be shown from the two observations: (1) sulfhydryl compounds such as cysteine and glutathione can completely or partially inhibit the SCEs induced by ascorbic acid, mitomycin C, adriamycin and maleic hydrazide; (2) the amounts of free radicals in root tips correlate with the frequencies of SCE in root tip cells.     

Demonstration of sister chromatid differentiation in human amniotic fluid cells after partial synchronization with BrdU or dT surplus

Summary Protocols are compared demonstrating sister chromatid differentiation (SCD) in human amniotic fluid (AF) cells with and without partial synchronization. Partial synchronization both with an excess of 5-bromodeoxyuridine (BrdU) and an excess of thymidine leads to an increase of metaphases with SCD. Compared with unsynchronized cells, the rate of sister chromatid exchanges (SCE) is not increased. Studies on the late replicating X chromosome of female cells showed that the addition of mitomycin C (MMC) after releasing the thymidine block preferentially induces SCEs in late replicating regions. The partial synchronization with thymidine surplus provides a good basis for SCE experiments with AF cells and facilitates the prenatal diagnosis of diseases characterized by changes in the SCE rate.     

Bromodeoxyuridine (BrdU) template and thymodine pool effects on high frequencies of sister-chromatid exchange (SCE) in Bloom syndrome cells and a mutant cell line (AsHa) originated from ataxia telangiectasia

The effect of bromodeoxyuridine (BrdU)-substituted DNA template and thymidine (dT) pool on excess sister-chromatid exchanges (SCEs) was studied in Bloom syndrome (BS) cells and an ataxia telangiectasia (AT)-derived mutant cell line (AsHa). When BS endomitotic cells were labeled with low and high (or high and low) BrdU concentrations during S₁ and S₂, only the BrdU concentration during S₁ phase affected the observed SCE. In BS cells about a 10-fold increase in SCEs occurs during or following replication on a BrdU-substituted template (high-high and high-low BrdU labeling) relative to the normal DNA template. SCEs decreased to about half in AsHa cells labeled with various BrdU doses (40, 60, 80 and 100 μg/ml) during only S₁, compared with those labeled during S₁ and S₂. Co-cultivation of AsHa and BS cells resulted in a significant reduction in SCE level from 70 to 13–17 in BS cells, lowered the BrdU concentrations necessary for sister-chromatid differential (SCD) staining from 40 to 10 μg/ml with normal SCE level and resulted in decreased level of SCEs at high BrdU concentrations (80–100 μg/ml) 12–14 SCE) in AsHa cells, compared with the originally increased SCE level (36.65 SCE at 100 μg/ml) without co-culture. However, co-cultivation between AsHa and normal cells lowered the BrdU dose necessary for SCD staining from 40 to 30 μg/ml; the dT pool possibly balanced at this level, which is clearly higher than that at co-cultivation between AsHa and BS cells. The reason for the very high BrdU doses needed to achieve SCD would seem to be that AsHa cells have high levels of thymidylate (TMP) synthetase, which maintain a large endogenous thymidine pool. This has been confirmed by direct measurement. These findings strongly support that excess and decreased dT pools are closely related to the condition necessary for high SCE induction.     

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.     

Spontaneous sister-chromatid exchange frequencies in human B and T lymphocytes at BrdU borderline concentrations for sister-chromatid differentiation

Human peripheral blood B and T lymphocytes, highly purified by immunologic methods, were supplemented with gamma-irradiated unseparated autologous mononuclear cells to restore helper functions and stimulated with pokeweed mitogen and phytohemagglutinin, respectively. Spontaneous sister-chromatid exchange (SCE) frequencies were investigated in proliferating B and T lymphocyte cultures labeled with the cell-type-specific borderline concentrations of 5-bromodeoxyuridine (BrdU) for sister-chromatid differentiation (SCD). B lymphocytes from 6 different donors showed mean values of 3.28-3.72 SCE events/cell. In T lymphocytes, mean values of 6.30-7.28 SCEs/cell were observed. The differences between the SCE distributions of the cell populations are highly significant. The results show that the differences in the spontaneous SCE frequencies between human B and T lymphocytes were not due to a difference in the uptake of BrdU.     

Differential BrdU uptake in 3 cell cycles and the resultant 3-way sister-chromatid differentiation at M3 endoreduplicated chromosome level--a hypothesis

The M3 endoreduplicated chromosomes account for SCE1-3 in a compact form after 3-way sister-chromatid differentiation (3-way SCD). However, a difficulty is faced in the analysis and interpretation of these results. Keeping this in view, the present work attempts to explain a number of possibilities correlating the SCD patterns to the probable patterns of uptake of bromodeoxyuridine (BrdU) over 3 successive cell cycles in M3 endoreduplicated chromosomes. This has been done to facilitate understanding of the staining patterns which could be obtained in the M3 endoreduplicated chromosomes after 3-way SCD, and further for the speedy analysis of such chromosomes, especially in scoring SCE1-3 precisely.     

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.     

Sister chromatid exchange in highly purified human B and T lymphocytes

Summary Sister chromatid exchange (SCE) rates were determined in human peripheral blood B and T lymphocyte populations highly purified by immunologic methods. The purified populations were supplemented with -irradiated unseparated autologous mononuclear cells to restore helper-functions and stimulated with pokeweed mitogen (PWM) and phytohemagglutinin (PHA), respectively. Measured at the different peaks of proliferation after identical bromodeoxyuridine (BrdU) incubation times, T lymphocytes showed significantly higher SCE frequencies than B lymphocytes. In both populations, different proliferation kinetics and a different minimal BrdU concentration for sister chromatid differentiation (SCD) were observed.     

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.     

Triethylene melamine-induced sister-chromatid exchange in murine lymphocytes exposed in vivo

The induction of sister-chromatid exchange (SCE) by triethylene melamine (TEM), a known animal carcinogen, was investigated in an in vivo exposure/in vitro culture murine lymphocyte assay. Dose-related increases in SCE were observed in B6D2F1 mice following a single i.p. injection of 0.5, 1 or 2 mg/kg TEM. SCE frequencies remained elevated over baseline levels at 24 h post exposure. It is hoped that studies of this nature can determine whether the in vivo/in vitro murine lymphocyte SCE assay is useful for predicting the carcinogenic potential of an agent.     

Analysis of spontaneous sister-chromatid exchanges in vivo by three-way differentiation

By applying an adaptation of the method of three-way differentiation to murine bone marrow cells in vivo, the basal frequency of sister-chromatid exchange (SCE) per cell was evaluated. An SCE frequency directly proportional to the estimated relative incorporation of 5-bromodeoxyuridine (BrdU) to the chromosomes was observed for the 3 consecutive cell cycles, implying that the majority, if not all, of the SCEs in vivo were produced by the incorporated BrdU. This conclusion was supported by the finding that in the first cycle of division, a very high frequency of cells without SCE was observed. From these data, a spontaneous frequency of SCE as low as 0.15 SCE/cell/cell cycle was inferred.     

Sister-chromatid exchanges and chromosomal aberrations in chick embryos after treatment with the fungicide maneb

The genotoxic potential of a commercial formulation of the fungicide maneb (Maneb 80, containing 80% manganese ethylenebisdithiocarbamate as active ingredient) in chick embryos was evaluated, using as genetic end-points the frequency of sister-chromatid exchanges (SCE) and of chromosomal aberrations. Unincubated eggs were dipped in 0, 0.5, 1.5, 4.5, 13.5 or 27 g/l maneb aqueous solutions for 30 sec. Eggs were then incubated for 4 days. Maneb significantly increased SCE values at 13.5 g/l and at 27 g/l (means 1.33 and 1.74) over the control value (mean 0.87). The concentration of 27 g/l, which corresponds to 10.8 times the recommended maximum application level for use in the field, also resulted in a high mortality rate. No clastogenic effects following exposure to maneb were observed.     

Effect of tetrandrine on micronucleus formation and sister-chromatid exchange in both in vitro and in vivo assays

The genotoxicity of tetrandrine, a drug potentially useful for the treatment of silicosis, was studied using the micronucleus and the sister-chromatid exchange (SCE) assay systems. Cultured Chinese hamster lung (V79) cells were used for the in vitro micronucleus and sister-chromatid exchange studies. Mouse bone marrow was used for the in vivo micronucleus assay and mouse spleen cells for the in vivo/in vitro sister-chromatid exchange analysis. The results show that SCE levels in V79 and in spleen cells were significantly elevated by treatment with tetrandrine at doses above 0.08 mg/ml and 100 mg/kg bw, respectively. Increased tetradrine-induced SCE in vitro was metabolic activation dependent. Tetrandrine failed to induce micronuclei at any of the doses tested. A decrease of replicative index with an increase in the concentration of tetrandrine was found both in vitro and in vivo. These results indicate that tetrandrine is a weak indirect-acting genotoxicant.