Cytotoxic effect and induction of sister chromatid exchange in exponentially growing rat 9L gliosarcoma cells after brief exposure to BrdU

The magnitude of DNA modulation in rat 9L gliosarcoma cells after a brief exposure to bromodeoxyuridine (BrdU) was studied by assaying colony-forming efficiency (CFE) and the number of sister chromatid exchanges (SCEs) per metaphase. The CFE assay showed that a 1-hr exposure to BrdU, at concentrations ranging from 10 to 1000 microM, produced a maximum cell kill of 5%. After a 2-hr exposure to 20 microM BrdU, the surviving fraction was 0.99, and even at a BrdU concentration of 1000 microM, 77% of the 9L cells survived. Compared with control cultures, the relative number of SCEs per metaphase in treated cultures was increased after a 1-hr exposure to BrdU at concentrations of 100 microM or more and after a 2-hr exposure to concentrations of 20 microM or more; no increase was observed in cells treated for 30 min with BrdU at concentrations up to 1000 microM. When the treated cells were allowed to grow in BrdU-free growth medium, the number of SCEs per metaphase returned to the control level within 24 hr, even after exposure to BrdU at concentrations as high as 1000 microM. These results demonstrate that exposure to BrdU at concentrations of up to 1000 microM for 30 min, 100 microM for 1 hr, and 20 microM for 2 hr causes little modulation of DNA.     

Mutagenic effects of brief exposure to bromodeoxyuridine on mouse FM3A cells

Abstract. The time- and dose-dependency of the mutagenic effects of bromodeoxyuridine (BrdU), a thymidine analogue used for cell kinetics studies in vivo and in vitro , were investigated in FM3A cells. Cells incubated with 50–1000 fin BrdU for 72 h showed some inhibition of growth. Cells cultured in BrdU-free medium for 3 d after a 30 min or 2 h exposure to BrdU showed no growth inhibition, while those previously exposed for 24 h to BrdU showed retarded growth. After a 30 min exposure, 60% of cells were labelled with BrdU; after 2 h 70%; and after 24 h almost 100%. After incubation in BrdU-free medium for 3 d (the time required for this cell line to express mutation), cells previously treated for 30 min or 2 h showed reduced BrdU positivity, whereas almost 100% of those treated for 24 h remained BrdU positive. The mutation rate, determined by the number of colonies resistant to ouabain (2 mM) and 6-thioguanine (10 μ) 3 d after exposure to BrdU, was not affected by a 30 min treatment with up to 1000 μ BrdU. Cells treated for 1 or 2 h showed increased resistance to ouabain after exposure to BrdU at concentrations above 100 μM; cells treated for 12 or 24 h showed an increased mutation rate at BrdU concentrations above 50 μM… The number of colonies resistant to 6-thioguanine did not increase in cells treated with BrdU at concentrations up to 1000 μM for 1, 12 or 24 h. We cannot conclude with certainty that brief exposure to BrdU does not modulate DNA to the point of mutation. This study may serve as a guideline for limiting the dose and time of exposure to BrdU for cell kinetics studies in vivo and in vitro.     

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.     

Influence of low doses of BrdU and estimation of spontaneous SCE in CHO chromosomes: three-way differential staining and an immunoperoxidase method

The influence of low doses of 5-bromodeoxyuridine (BrdU) on the occurrence of sister chromatid exchanges (SCEs) during the first cell cycle, when unsubstituted DNA templates replicate in the presence of the halogenated nucleoside (SCE1) has been assessed in third mitosis (M3) Chinese hamster ovary (CHO) cells showing three-way differential (TWD) staining. In addition, lower concentrations of BrdU, not detectable by Giemsa staining, have been tested by a high resolution immunoperoxidase method (anti-BrdU monoclonal antibody) and SCEs were scored in second mitosis (M2) cells. Our findings was a dose-response curve for SCE1 that allows an estimated mean spontaneous yield of 1.32/cell per cell cycle by extrapolation to zero concentration of BrdU. On the other hand, when the total SCE frequency corresponding to the first and second rounds of replication (SCE1+SCE2) found in M3 chromosomes was compared with the yield of SCEs scored in M2 cells grown in BrdU at doses lower than 1 M no further reduction was achieved. This seems to indicate that SCEs can occur spontaneously in this cell line, though the estimated frequency is higher than that reported in vivo.by S. Wolff     

Human colorectal tumours in short-term organ culture A stathmokinetic study

Abstract. Short-term organ culture, using a technique to preserve epithelial/stromal interaction and metabolism, is a useful technique for carrying out kinetic studies on human colorectal carcinoma and adjacent normal mucosa, providing initial perturbations of proliferative indices are allowed to settle. Tumours require 3.0 μg/ml vincristine for complete metaphase arrest compared with mucosa, which needs 0.5 μg/ml, a 6-fold difference. Using a stathmokinetic technique, the birth rate of tumour cells is 10.21 cells/1000 cells per hr, compared with 7.73 cells/1000 cells per hr for mucosa, a statistically significant difference ( P < 0.01).     

Sister chromatid exchanges in Allium cepa

Differential staining of sister chromatids with Giemsa after BrdU incorporation into DNA was performed in Allium cepa L. chromosomes. A treatment solution containing 10^–7 M FdU, 10^–4 M BrdU and 10^–6 M Urd was found to ensure BrdU incorporation without affecting cell cycle duration. After several procedures before staining the slides with Giemsa had been tested, treatment with the fluorochrome compound 33258 Hoechst, exposure to UV light and heating at 55° C in 0.5×SSC, were found to be essential for good differentiation. The distribution of SCEs per chromosome agrees with the expected Poisson distribution. The mean value of SCEs per chromosome occurring when cells were exposed to the treatment solution for two consecutive rounds of replication (=5.5) was double the mean value observed when cells were exposed to the same treatment for only one round of replication (=2.8). SCEs were found to occur more frequently in those chromosome regions corresponding neither to C-bands nor to late replicating DNA-rich regions. Finally, the occurrence of SCEs involving less than the width of a chromatid is discussed.     

Granuloma pouch assay IV. Induction of sister-chromatid exchanges in vivo

SCEs were induced, in vivo, in cells of a rapidly proliferating subcutaneous granulation tissue, initiated by the formation of a subcutaneous air pouch, on the backs of adult male rats (Granuloma Pouch Assay). 2 days after pouch formation the test compounds were applied, and 24 h later the granulation tissue was excised and dissociated into single cells. Isolated cells were cultured in vitro in media containing BrdU, and SCEs were determined within 24–48 h. The spontaneous frequency was 14.4±1.1 per metaphase.Mitomycin C (MMC) and cyclophosphamide (CP) induced a significant and dose-dependent increase in SCE frequencies. Results obtained after i.v., i.p. and intra-pouch application routes are compared.     

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.     

Frequency of sister-chromatid exchanges depending on the amount of 5-bromodeoxyuridine incorporated into parental DNA

Chinese hamster D-6 cells were grown for two cell cycles. The effect of 5-bromodeoxyuridine (BrdU) on the frequencies of sister-chromatid exchanges (SCEs) in these cells was investigated by the BrdU-labeling method. A low concentration (5 μM) of BrdU was inoculated in the first cell cycle for SCE counting. When excess concentrations (100–1000 μM) of BrdU were added subsequently in the second cell cycle, a 1–2-fold increase of SCE frequencies was observed. When excess thymidine (dT) (100–1000 μM) was supplied instead of BrdU, the incidence of SCE also increased. When cells were exposed to high concentrations (50–200 μM) of BrdU in the first cell cycle, a 1–4-fold increase in SCE frequencies was observed. This incidence of SCE was largely dependent on the concentration of BrdU and dT used in the second cell cycle. These results suggest that efficient SCE induction by BrdU is related to the BrdU residue incorporated into parental DNA strands.     

Induction of sister-chromatid exchanges in Chinese hamster ovary cells by the biotic ketoaldehyde methylglyoxal

The number of sister-chromatid exchanges (SCEs) per metaphase was determined in Chinese hamster ovary cells after 16 h exposure to methylglyoxal (MG) concentrations ranging from 0.1 to 0.75 mM. MG produced an increase of SCE frequency that proved to be dose-dependent, and to reach a maximum of 2 X baseline at the highest nontoxic concentration (0.5 mM).     

Cell Cycle Kinetics of Aerated, Hypoxic and Re-Aerated Cells In Vitro Using Flow Cytometric Determination of Cellular Dna and Incorporated Bromodeoxyuridine

Abstract. The effects of extreme hypoxia on cell cycle progression were studied by simultaneous determination of DNA and bromodeoxyuridine (BrdU) contents of individual cells. V79-379A cells were pulse-labelled with BrdU (1 μM, 20 min, 37°C) and then incubated for up to 12 hr in BrdU-free medium under either aerated or extremely hypoxic conditions. After the incubation interval (0-12 hr), the cells were trypsinized and fixed in 50% EtOH. Propidium iodide and a fluorescein-labelled monoclonal antibody to BrdU were then used to quantify DNA content and incorporated BrdU, respectively. Measurements in individual cells were made by simultaneous detection of green and red fluorescence upon excitation at 488 nm using flow cytometry. Bivariate analysis revealed progression of BrdU-labelled cells in aerated cultures out of S phase, into G₂ and cell division, with halving of mean fluorescence, and back into S phase by approximately 9 hr after the BrdU pulse. Hypoxia immediately arrested cells in all phases of the cell cycle. Both the DNA distribution and the bivariate profile of cells that were fixed from 2 to 12 hr after induction of hypoxia were identical to the 0 hr controls. the percent of cells with green fluorescence in a mid-S phase window remained 100% and the mean fluorescence of these cells remained at control (0 hr) levels. This indicates that, under hypoxic conditions, cells were moving neither into nor out of S phase. Cultures that had been hypoxic for 12 hr exhibited an increasing rate of BrdU uptake with time after re-aeration. Re-aerated cells were able to complete or initiate DNA synthesis, but their rates of progression through the cell cycle were markedly reduced. A large fraction of cells appeared unable to divide up to 12 hr following release from hypoxia.     

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.     

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.     

Physical,chemical, and biological factors affecting sister-chromatid exchange induction in human lymphocytes exposed to mitomycin C prior to culture

In experiments to assess the effects of several biological, chemical, and physical variables on sister-chromatid exchange (SCE) induction in cultured lymphocytes exposed to mitomycin C (MMC) before PHA stimulation we observed: (1) high SCE frequencies in female cells, and normal SCE frequencies in Y-bearing metaphases in mixed cultures containing equal numbers of MMC-treated female lymphocytes and untreated male lymphocytes; (2) small, but statistically significant, decreases in SCEs with increasing pH after G₀ exposure in the pH range 6.6–7.6; (3) pronounced reductions in MMC-induced SCEs in lymphocytes exposed at 4°C vs. 37°C. In other studies, SCE induction was evaluated in cultures exposed during G₀ to MMC concentrations ranging from 0.25 to 2.5 μg/ml for varying time intervals ranging from 5 min to 24 h. For all concentrations tested SCE induction varied as a linear function of G₀ exposure time. To compare SCE induction between cultures, we calculated the mean frequencies of SCEs induced per metaphase/unit dose MMC/unit G₀ exposure time (SCE/μg/h). A mean frequency of 20.7 ± 4.8 SCE/μg/h was observed for 41 lymphocyte cultures suggesting that a single term adequately describes the rate of SCE induction following G₀ exposure to a 10-fold range in concentration of MMC for time intervals of 30 min to 24 h.     

Chromosome aberrations, micronuclei and sister-chromatid exchanges (SCEs) in rat liver induced in vivo by hepatocarcinogens including heterocyclic amines

The induction of chromosome aberrations, micronuclei and SCEs was studied in hepatocytes of F344 rats exposed in vivo to hepatocarcinogens. Hepatocytes were isolated and allowed to proliferate in Williams' medium E supplemented with epidermal growth factor. Cells were fixed after a culture period of 48 h. Oral administration of dimethylnitrosamine at doses of 2.5-20 mg/kg body weight (bw) induced (1) chromosome aberrations in up to 27% of the metaphase cells 2-48 h after its administration, (2) SCEs with a frequency of up to 0.9 per chromosome 2-48 h after its administration, and (3) micronuclei in up to 2.9% of the cells 16-48 h after its administration. Oral administration of 2-acetylaminofluorene at doses of 6.25-200 mg/kg bw induced (1) chromosome aberrations in up to 35% of the metaphase cells after 2-48 h, (2) SCEs at up to 0.9 per chromosome and (3) micronuclei in up to 2.5% of the cells with a maximum after 4 h. Oral administration of CCl4, a non-genotoxic hepatocarcinogen, at a dose of 1600 mg/kg bw did not induce chromosome aberrations, SCEs or micronuclei within 4-72 h. Intraperitoneal injections of Trp-P-1, Glu-P-1, MeIQx, IQ and nitro-IQ resulted in chromosome aberrations in up to 16% of the metaphase cells and SCEs at up to 0.9 per chromosome, while injections of Trp-P-2 and Glu-P-2 produced SCEs at up to 0.7 and 1.1 per chromosome, respectively. The present method of in vivo cytogenetic assay using rats without partial hepatectomy or mitogen treatment in vivo should be useful for evaluating the tumor-initiating activities of hepatocarcinogens.     

Sequential study on the influence of adriamycin on cell proliferation and ultrastructure of cultured mammary tumor cells

The time-dependent cytocidal and growth inhibitory effects of Adriamycin (ADM) on monolayer cultures of 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumor cells were analyzed. The inhibitory effect on cell proliferation was assessed by colony formation in soft agar. Growth inhibition and [3H]thymidine labeling indices clearly demonstrate a dose-dependent antimitotic and cytotoxic effect of the drug. At low concentrations (10(-9)-10(-8) M), 90-100% of cells survived 24-hr exposure. At a higher concentration (10(-5) M), 75-80% of cells survived after 8-hr exposure; by 72 hr only 20-30% of the cells remained. Autoradiographic examination of the pulse-labeled cultures demonstrated no change in the proportion of cells in S-phase during the first 4 hr of treatment. Subsequently DNA synthesis was completely abolished and remained inhibited for the duration of the experiment (72 hr). Clonogenic assay revealed a complete arrest of growth in cells exposed to 10(-5) M ADM and greater than 60% inhibition of cell proliferation at 10(-7) M. Ultrastructural changes were not observed in cells during the first 4 hr of treatment; however, after 8 hr most surviving cells exhibited alterations in nuclear chromatin. The surviving cells showed mitochondrial degeneration, myelin body formation, and vacuolization of the endoplasmic reticulum. This study shows the potential usefulness of the primary culture system in drug evaluation. In addition, serial observation of the effects of ADM revealed a cell subpopulation of the primary culture with differential sensitivity to the drug.     

SOME OBSERVATIONS OF ABNORMAL EMBRYOS INDUCED BY SHORT-PERIOD TREATMENT WITH CHLORAMPHENICOL DURING EARLY DEVELOPMENT OF SEA URCHIN

Sea urchin embryos, which were treated with 5 × 10⁻³ M chloramphenicol for 1 to 4 hr at certain stages before hatching, developed to several types of abnormal embryos. No significant effect on the shape of the embryo was observed when the concentrations of chloramphenicol used in the short-period treatment were lower than 2 × 10⁻³ M. Embryos up to the 2-cell stage, treated with 5 × 10⁻³ M chloramphenicol for a short period, became small blastulae filled with mesenchyme-like cells (type A). A similar effect of puromycin (2 μg/ml) was also observed at this stage. When the chloramphenicol treatment (for 1 to 4 hr) was applied at 8 ∽ 32-cell stages, vegetalized larvae were produced (type B). Embryos treated with chloramphenicol at 7 hr after insemination at 20°C, developed to another type of abnormal larva different from the previous types (type C). A concentration of puromycin (2 μg/ml) which inhibited protein synthesis to the same degree as 5 × 10⁻³ M chloramphenicol, induced only type A. Between these chloramphenicol-sensitive stages, there were chloramphenicol-insensitive stages for forming abnormal embryos.     

Diurnal variations in serum testosterone concentrations of captive adult male chacma baboons (Papio ursinus)

Captive adult male chacma baboons (Papio ursinus) housed with natural lighting exposure and blood sampled at 3-hr intervals showed significant diurnal variations in serum testosterone concentrations. Low mean concentrations were found at 0800 hr approximately 1 hr after sunrise and mean concentrations were their highest at 2000 hr approximately 1 ¼ hr after sunset.     

In vivo BrdU-33258 Hoechst analysis of DNA replication kinetics and sister chromatid exchange formation in mouse somatic and meiotic cells

BrdU (5-bromodeoxyuridine)-33258 Hoechst methods have been adapted for in vivo analyses of replication kinetics, sister chromatid differentiation and sister chromatid exchange (SCE) formation in mice. Sufficient in vivo BrdU substitution for cytological detection was effected with multiple intraperitoneal injections of the analogue. The combination of centromere staining asymmetry and sister chromatid differentiation at metaphase permits unambiguous determination of the number of replications in BrdU and dT (deoxythymidine) undergone by individual cells. Late-replicating regions in marrow and spermatogonial chromosomes are highlighted by bright fluorescence after sequential incorporation of BrdU followed by dT during a single DNA synthesis period. SCEs are analyzed in marrow and spermatogonial metaphases after successive complete cycles of BrdU and dT incorporation. Significant induction of SCE was observed with both mitomycin C and cyclophosphamide; the latter drug requires host-mediated activation to be effective. In meiotic metaphase cells harvested two weeks after BrdU incorporation, satellite DNA asymmetry, sister chromatid differentiation and SCE could be detected in a few chromosomes, most frequently the X and the Y.     

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.