A unique human mutant B-lymphoblastoid cell line (ataxia telangiectasia) which exhibits increased siste-chromatid exchange retaining hypersensitivity to neocarzinostatin and bleomycin

Chromosome aberrations and sister-chromatid exchanges (SCEs) were examined in 4 ataxia telangiectasia (AT)-derived B-lymphoblastoid cell lines (B-LCLs) (AT-S, AT-SHI, AT-SHI B13A and AsHa) following treatments with neocarzinostatin (NCS) and bleomycin. All of these cell lines exhibited extremely high frequencies of chromosome aberrations with the NCS and bleomycin treatments. Among them, AsHa, a mutant B-LCL originating from an AT patient, showed high frequencies of SCEs under high bromodeoxyuridine (BrdU) concentrations retaining hypersensitivity to NCS and bleomycin with regard to chromosome aberrations. A clear BrdU dose-dependent increase in SCEs (9.85 SCEs/cell at 40 μg/ml, 36.65 SCEs/cell at 100 μg/ml on average) in this mutant was observed. When AsHa mutant cells were treated with NCS (0.02 μg/ml) and/or bleomycin (5.0 μg/ml) under 40 μg/ml BrdU (minimum BrdU concentration for sister-chromatid differential staining), SCE levels increased from 9.85 (baseline level) to 21.1 with NCS and 20.5 with bleomycin, in a dose-dependent manner. These observations indicate that AsHa is a unique AT-derived mutant cell clone with a high SCE character retaining the original hypersensitivity to bleomycin and NCS.     

Sister chromatid exchanges induced by sarcolysine in human lymphocytes in vitro

The effect of three concentrations of sarcolysine (0.5 micrograms/ml, 1 microgram/ml and 2 micrograms/ml) on the sister chromatid exchanges (SCE) was investigated in human lymphocytes in vitro. A dose related increase in SCEs frequencies was observed after sarcolysine administration and also a delayed development of cell cycle has been induced by the two last concentrations. The variation range of SCEs per cell was dose-dependent and it was considered to represent the acquired genetic instability induced by the drug.     

Cytogenetic effects of penicillamine

Penicillamine (PA), a drug used for the treatment of rheumatoid arthritis induces sister-chromatid exchanges (SCEs) and chromosome aberrations in cultivated mammalian cells. PA in concentrations from 400 micrograms/ml upward induced SCEs and proliferative delay in human blood cultures when added for the last 24 h of the culture period. In V79 Chinese hamster cells SCE induction was found after acute exposure to PA before the addition of BrdUrd and after chronic exposure during one cell cycle in the presence of BrdUrd. The effect of PA on SCE frequencies occurred both after treatment in complete medium and in serum-free medium and was not influenced by the application of an S9 mix. The simultaneous addition of peroxidase reduced the PA-induced SCEs whereas catalase did not show any effect. Chromosome analysis in the first mitosis after PA treatment revealed a significant increase in the incidence of chromosome aberrations and endoreduplication. The results are discussed with respect to the cause and the significance of the observed effects in connection with mutagenicity testing.     

Effects of theophylline on chromosomal breakage and sister-chromatid exchange

Frequencies of both sister-chromatid exchange (SCE) and chromosomal breakage (CB) were studied in the lymphocytes of normal individuals (10 and 7 individuals respectively). The cells were exposed in vitro to 3 different concentrations of theophylline (1, 10 and 100 micrograms/ml). A significant concentration effect of the drug was demonstrated for both SCEs and CB. Utilizing a Dunnett's test for individual comparisons, the 10 and 100 micrograms/ml concentrations both demonstrated a significant elevation of SCEs and CB compared to the untreated control cultures. This study suggests that in vitro concentrations of theophylline equal to or greater than 10 micrograms/ml, corresponding to serum levels attained during therapy, increase the frequency of SCEs and chromosome breakage in human lymphocytes.     

Induction of chromosome aberrations and sister-chromatid exchanges in CHO-K1 cells by o-phenylphenol

o-Phenylphenol (OPP), is used in Japan as a fungicide in food additives for citrus fruits. The induction of chromosome aberrations and sister-chromatid exchanges (SCEs) by OPP in cultured Chinese hamster ovary (CHO-K1) cells was studied. Cells were exposed to various concentrations of OPP ranging from 50 to 175 micrograms/ml for 3 h, and further incubated for 27 and 42 h. These incubation periods are almost equal to 2 and 3 cell cycles. SCEs and chromosome aberrations were induced by OPP at concentrations of 100, 125 and 150 micrograms/ml after the incubation for 27 h. For chromosome aberrations, chromatid breaks and exchanges there was a dose-dependent increase. Diplochromosomes due to endoreduplication were also caused by the same concentrations of OPP in a dose-dependent manner. After incubation for 42 h, chromosome aberrations were also increased by OPP at concentrations of 100 and 125 micrograms/ml, but the frequencies of SCEs were not significantly different from those of the control. These results suggest that OPP has a cytogenetic toxicity, and that the DNA damage resulting in SCEs induced by OPP is relatively short-lived and can be repaired during the longer incubation time.     

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.     

Presence of a Base Line Level of Sister Chromatid Exchanges in Somatic Cells of DROSOPHILA MELANOGASTER IN VIVO and IN VITRO

Sister chromatid exchanges (SCEs) under in vivo and in vitro conditions were examined in ganglion cells of third-instar larvae of Drosophila melanogaster (Oregon-R). In the in vivo experiment, third-instar larvae were fed on synthetic media containing 5-bromo-2'-deoxyuridine (BrdUrd). After two cell cycles, ganglia were dissected and treated with colchicine. In the in vitro experiment, the ganglia were also incubated in media containing BrdUrd for two cell cycles, and treated with colchicine. SCEs were scored in metaphase stained with Hoechst 33258 plus Giemsa. The frequencies of SCEs stayed constant in the range of 25-150 micrograms/ml and 0.25-2.5 micrograms/ml of BrdUrd in vivo and in vitro, respectively. SCEs gradually increased at higher concentrations, strongly suggesting that at least a fraction of the detected SCEs are spontaneous. The constant levels of SCE frequency were estimated, on the average, at 0.103 per cell per two cell cycles for females and 0.101 for males in vivo and at 0.096 for females and 0.091 for males in vitro. No difference was found in the SCE frequency between sexes at any of the BrdUrd concentrations. The analysis for the distribution of SCEs within chromosomes revealed an extraordinarily high proportion of the SCEs at the junctions between euchromatin and heterochromatin; the remaining SCEs were preferentially localized in the euchromatic regions of the chromosomes and in the heterochromatic Y chromosome. These results were largely inconsistent with those of Gatti et al. (1979).     

Lack of Spontaneous Sister Chromatid Exchanges in Somatic Cells of DROSOPHILA MELANOGASTER

Neural ganglia of wild type third-instar larvae of Drosophila melanogaster were incubated for 13 hours at various concentrations of BUdR (1, 3, 9, 27 micrograms/ml). Metaphases were collected with colchicine, stained with Hoechst 33258, and scored under a fluorescence microscope. Metaphases in which the sister chromatids were clearly differentiated were scored for the presence of sister-chromatid exchanges (SCEs). At the lowest concentration of BUdR (1 microgram/ml), no SCEs were observed in either male or female neuroblasts. The SCEs were found at the higher concentrations of BUdR (3, 9, And 27 micrograms/ml) and with a greater frequency in females than in males. Therefore SCEs are not a spontaneous phenomenon in D. melanogaster, but are induced by BUdR incorporated in the DNA. A striking nonrandomness was found in the distribution of SCEs along the chromosomes. More than a third of the SCEs were clustered in the junctions between euchromatin and heterochromatin. The remaining SCEs were preferentially localized within the heterochromatic regions of the X chromosome and the autosomes and primarily on the entirely heterochromatic Y chromosome.--In order to find an alternative way of measuring the frequency of SCEs in the Drosophila neuroblasts, the occurrence of double dicentric rings was studied in two stocks carrying monocentric ring-X chromosomes. One ring chromosome, C(1)TR94--2, shows a rate of dicentric ring formation corresponding to the frequency of SCEs observed in the BUdR-labelled rod chromosomes. The other ring studied, R(1)2, exhibits a frequency of SCEs higher than that observed with both C(1) TR94--2 and rod chromosomes.     

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.     

Analysis of low concentrations of 5-bromo-2-deoxyuridine on sister chromatid exchanges in human lymphocytes

To determine a concentration of 5-bromo-2-deoxyuridine (BrdU) sufficient for sister chromatid differentiation (SCD), and yet having a minimal effect on the number of sister chromatid exchanges (SCEs), we assessed the effect produced on the number of SCEs by low concentrations (1, 3, and 10 micrograms/mL) of BrdU. SCD was not obtained in 19% of the 31 subjects with 1 microgram/mL of BrdU, while the differentiation was adequate for all samples treated with 3 and 10 micrograms/mL. We statistically analysed the effects of these three different doses and found no significant difference in the number of SCEs obtained with the doses of 1 and 3 micrograms/mL, but a significant difference was observed between these two concentrations and 10 micrograms/mL. We therefore suggest that the dose of 3 micrograms/mL, while sufficient to produce reliable differential staining, still permits an adequate evaluation of the base line of SCEs and appears to enhance the sensitivity of the test to evaluate between-individual variations. Our experiments also underline that SCE counts should include the centromere exchanges.     

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

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

Tea tannin components modify the induction of sister-chromatid exchanges and chromosome aberrations in mutagen-treated cultured mammalian cells and mice

The modifying effects of tannin components extracted from green tea and black tea on mutagen-induced SCEs and chromosome aberrations were studied. These tannin components did not affect spontaneous SCEs and chromosome aberrations in cultured Chinese hamster cells. The frequency of SCEs and chromosome aberrations induced by mitomycin C (MMC) or UV was enhanced by the posttreatment with tea tannin components. When cells were post-treated with tea tannin components in the presence of metabolic enzymes of rat liver (S9 mix), the modifying effects on the induction of SCEs and chromosome aberrations by mutagens were complicated. MMC- and UV-induced SCEs and chromosome aberrations were suppressed by the posttreatment with tea tannin components at low concentrations (less than or equal to 6.7 micrograms/ml) with S9 mix. At a high concentration of tea tannin components (20 micrograms/ml) with S9 mix, a co-mutagenic effect was observed. The modifying effects of tea tannin components were shown to occur in the G1 phase of the cell cycle. In cells from a patient with xeroderma pigmentosum (XP) and a normal human embryo, MMC-induced SCEs were suppressed by the posttreatment with tea tannin components in the presence of S9 mix, and enhanced in the absence of S9 mix. On the other hand, tea tannin components modified SCE frequencies in UV-irradiated normal human cells but not in UV-irradiated XP cells. Our results suggested that tea tannin components themselves inhibited DNA-excision repair and resulted in a co-mutagenic effect, while in the presence of S9 mix metabolites of tea tannin components promoted DNA-excision repair activity and resulted in an antimutagenic effect. MMC-induced chromosome aberrations in mouse bone marrow cells were suppressed by the pretreatment with green tea and black tea tannin mixture.     

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).     

Developmental and cytogenetic effects of caffeine on mouse blastocysts, alone or in combination with benzo(a)pyrene

Mouse blastocysts were treated with caffeine and/or benzo(a)pyrene (BP), and the effects on development and on induction of sister chromatid exchanges (SCEs) were examined. Caffeine interfered with blastocyst development in a dose-related manner. At 4 mM, the highest concentration tested, caffeine interfered with development of blastocysts to all four endpoints: hatching, trophoblast outgrowth, inner cell mass (ICM) growth, and two-layer (primary endoderm and ectoderm) differentiation of ICMs. At 2 mM, caffeine reduced the incidence of both ICM growth and differentiation but did not affect hatching or formation of trophoblast outgrowths. At 1 mM, caffeine interfered only with ICM differentiation. Cell proliferation was least sensitive to caffeine and was reduced at concentrations of greater than or equal to 2 mM. Induction of SCEs was most sensitive to caffeine exposure; an increase in SCE frequency was observed at 0.1 and 0.5 mM. When caffeine was added to cultures with BP (1 microM, a concentration that was not embryotoxic and did not induce SCEs), both embryotoxic effects and SCE frequency were increased. The enhancing effect on SCE induction was particularly marked; as little as 0.1 mM caffeine was sufficient to cause doubling of induced SCE frequencies when added to cultures with BP.     

SCE frequencies in rabbit lymphocytes as a function of time after an acute dose of cyclophosphamide

Following acute and chronic exposures to various chemicals in vivo, the average SCE frequency in human and rabbit lymphocytes has generally been shown to decrease with time posttreatment. The rate of this decline varies, however, and little data have been published pertaining to the decrease in SCEs soon exposure. To gain more information about the immediate decline in SCEs with time, we injected rabbits with a single dose of 35 mg/kg cyclophosphamide (CP) and determined SCE levels in circulating lymphocytes at various times 5 h to 2 weeks after treatment. We observed a rapid decline in SCE frequencies within 5 days, and by 10 days post-exposure the SCE levels were back to control values. The distributions of SCEs among cells and the number of circulating lymphocytes were also analyzed at each time. Within 2–3 days posttreatment we observed a rapid loss of cells with high SCE levels concomitantly with a rapid decline in circulating lymphocytes and a decrease in the average SCE frequency. When the number of lymphocytes began to increase, the number of cells with normal SCE values also increased. By 10–11 days after CP, the lymphocyte count had recovered, the SCE frequency had returned to control levels, and the distribution of SCEs among cells was almost identical to the control distribution. These data, in addition to published information on rabbit lymphocyte lifespan, suggest that the decline in SCE levels with time posttreatment is a function of lymphocyte turnover.     

Bimodal distribution of sensitivity to SCE induction by diepoxybutane in human lymphocytes. I. Correlation with chromosomal aberrations

We carried out a cross-sectional analysis of sister-chromatid exchanges (SCEs) and chromosomal aberrations induced by diepoxybutane (DEB) in lymphocyte cultures from 58 normal blood donors. DEB-induced SCE frequencies were measured in all subjects and chromosomal aberrations in 18. Analysis of variance was used to assess the contributions of exposure to organic solvents, age, smoking history, alcohol and coffee consumption, and red and white blood cell counts to variations in DEB-induced SCEs. In 10 individuals, the epoxide-detoxifying enzyme, glutathione (GSH)-S-transferase mu, was also measured. We observed a bimodal distribution of DEB-induced SCEs in the study population. Approx. 24% of the individuals were twice as sensitive to the induction of SCEs by DEB as the remaining 76%. Lymphocytes from persons sensitive to SCE induction by DEB contained a 4.4-fold increase in the number of DEB-induced chromatid deletions and exchanges. Within sensitive and resistant groups, significant interindividual variations in DEB-induced SCE frequencies were noted. Cigarette smoking was weakly associated with lower SCE frequencies within each group. Genetic deficiency in GSH-S-transferase mu was not correlated with increased sensitivity to SCE induction by DEB. Sensitivity to induction of SCEs by DEB can be rapidly determined and may be a marker of sensitivity to the induction of genotoxicity by certain classes of mutagens.     

Modulating effects of naturally occurring indoles on SCE induction depend largely on the type of mutagen

The modulating effects of pretreatment of cultured cells with indole-3-carbinol (I3C) and indole-3-acetonitrile (I3A) on the induction of sister-chromatid exchanges (SCEs) by mutagens from different chemical classes were investigated. Cultured primary chick embryo hepatocytes were treated for different periods with I3C (25 micrograms/ml) and with I3A (35 micrograms/ml). Treatment with I3C resulted in a 3-fold increase in ethoxyresorufine-O-deethylase (Erod) activity and a 2-fold increase in ethoxycoumarine-O-deethylase (Etco) activity. Treatment with I3A resulted in a 1.6-fold increase in Erod activity and a 2-fold increase in Etco activity. Pretreatment of cultured primary chick embryo hepatocytes with I3C resulted in a 30-45% decrease in the number of SCEs induced by benzo[a]pyrene (B(a)P) and dimethylnitrosamine (DMN) in co-cultured V79 Chinese hamster cells. No decrease in SCE induction was observed for 2-aminoanthracene (2AA) and the direct-acting alkylating agent ethyl methanesulphonate (EMS). In contrast, when dibromoethane (DBE) was tested pretreatment with I3C resulted in an increase in SCE induction. Pretreatment with I3A again resulted in a 20-40% decrease in SCE induction for B(a)P whereas no decrease was observed for DMN, 2AA and EMS. The results of this study indicate that the type of effect of indole pretreatment largely depends on the type of mutagen selected.     

Cytotoxicity of tin on human peripheral lymphocytes in vitro.

The comparative effects of inorganic and organic tin compounds on chromosomes were assessed in human peripheral blood lymphocytes of healthy donors 20-40 years of age. The endpoints observed were chromosomal abnormalities, sister-chromatid exchanges (SCEs) and cell cycle kinetics. The maximum concentrations which reduced the replicative index by about 50%, of stannic chloride and trimethyltin chloride were 40 micrograms and 2 micrograms per culture respectively. The tested doses were 20 micrograms and 10 micrograms of stannic chloride and 1 microgram and 0.5 microgram of trimethyltin chloride. Both doses of stannic chloride induced a much higher frequency of chromosomal abnormalities (P less than 0.05-P less than 0.001) and a greater reduction of cell cycle kinetics than the corresponding relative doses of trimethyltin chloride. The frequencies of SCEs/cell induced by the latter were, however, slightly higher than those induced by the former.     

Effect of high-density extremely low frequency magnetic field on sister chromatid exchanges in mouse m5S cells

The induction of sister chromatid exchanges (SCEs) was evaluated in the cultured mouse m5S cells after exposure to extremely low frequency magnetic field (ELFMF; 5, 50 and 400 mT). Exposure to 5 mT and 50 mT ELFMF led to a very small increase in the frequency of SCEs, but no significant difference was observed between exposed and unexposed control cells. The cells exposed to 400 mT ELFMF exhibited a significant elevation of the SCE frequencies. There was no significant difference between data from treatments with mitomycin-C (MMC) alone and from combined treatments of MMC plus ELFMF (400 mT) at any MMC concentrations from 4 to 40 nM. These results suggest that exposure to highest-density ELFMF of 400 mT may induce DNA damage, resulting in an elevation of the SCE frequencies. We suppose that there may be a threshold for the elevation of the SCE frequencies, that is at least over the magnetic density of 50 mT.     

Effect of high-density extremely low frequency magnetic field on sister chromatid exchanges in mouse m5S cells

The induction of sister chromatid exchanges (SCEs) was evaluated in the cultured mouse m5S cells after exposure to extremely low frequency magnetic field (ELFMF; 5, 50 and 400 mT). Exposure to 5 mT and 50 mT ELFMF led to a very small increase in the frequency of SCEs, but no significant difference was observed between exposed and unexposed control cells. The cells exposed to 400 mT ELFMF exhibited a significant elevation of the SCE frequencies. There was no significant difference between data from treatments with mitomycin-C (MMC) alone and from combined treatments of MMC plus ELFMF (400 mT) at any MMC concentrations from 4 to 40 nM. These results suggest that exposure to highest-density ELFMF of 400 mT may induce DNA damage, resulting in an elevation of the SCE frequencies. We suppose that there may be a threshold for the elevation of the SCE frequencies, that is at least over the magnetic density of 50 mT.