Effect of Free Radicals and Temperature on Sister Chromatid Exchanges in Hordeum vulgare L.

The clastogenic (chromosome-damaging) effect of many chemical and physical agents is believed to be mediated by reactive oxygen-detived radicals. The interaction of these free radicals with DNA and the significance of the radical-induced DNA lesions in mutagenesis and carcinogenesis have been the subjects of increasing interest during recent years. Sister chromatid exchange (SCE) reflects an interchange between DNA molecules at homologous loci within a replicating chromosome. SCE analysis was found to have increased use for monitoring the exposure of cell to mutagenic carcinogens. The authors found that the induction of SCEs in cells of Hordeum vulgare L. by ascorbic acid, mitomycin C, adriamycin and maleic hydrazid was through the action of free radicals. They also studied the influence of growth temperature on average generation time(AGT) and SCEs. and disclosed a close correlation between AGT and SCEs. The Brdu-Giemsa techniques were used for the detection of SCEs and AGT in cytological preparations of metaphase chromosomes.     

Effects of amino acids on sister-chromatid exchanges.

The effects of 10 amino acids on sister-chromatid exchange (SCE) frequency in human peripheral blood lymphocytes (PBL) and six amino acids on the SCE frequency in root tip cells of Hordeum vulgare were studied. Alanine (Ala), glycine (Gly), phenylalanine (Phe), valine (Val), histidine (His) and serine (Ser) induced a significant increase in SCE in PBL but threonine (Thr), isoleucine (Ile), lysine (Lys) and arginine (Arg) did not. Ala, Gly, Thr, Ile and Val induced a significant increase in SCE in root tip cells of Hordeum vulgare but Lys did not. The effect of Lys and bromodeoxyuridine (BrdU) on SCE levels in PBL and the interaction between them were also studied. The results show that Lys can inhibit the SCE induced by BrdU.     

DNA crosslinking, sister-chromatid exchange and specific-locus mutations

Chinese hamster ovary cells were treated with the DNA-crosslinking chemicals, mitomycin C (MMC) and porfiromycin (POR), and their monofunctional derivative decarbamoyl mitomycin C (DCMMC). After exposure, the cells were studied for the induction of sister-chromatid exchanges (SCEs) and mutations at the hypoxanthine phosphoribosyltransferase and adenine phosphoribosyltransferase loci. The frequency of SCEs varied significantly in successive sampling intervals, requiring the weighting of each interval by the percentage of second-division mitosis in that interval to obtain the mean SCE frequency for each dose. All 3 compounds were potent inducers of SCEs but weakly mutagenic. All 3 chemicals by concentration were approximately equally effective in inducing SCEs or mutations. When the induced SCEs and mutations were compared at equal levels of survival, DCMMC was slightly more effective than MMC or POR in inducing SCEs and somewhat less mutagenic. These results indicate that the DNA interstrand crosslink is not the major lesion responsible for the induction of SCE or mutation by these compounds.     

Effects of temperature on chemically induced sister-chromatid exchange in human lymphocytes

Lymphocytes from healthy adults were studied for sister-chromatid exchanges (SCEs) when pulse-treated in G0 with mitomycin C (MMC), ethyl methanesulfonate (EMS), or 4-nitroquinoline N-oxide (4NQO) at various temperatures ranging from 0 degrees C to 41 degrees C and then cultured in medium containing 5-bromodeoxyuridine at 37 degrees C. The results showed that the frequencies of SCEs induced by MMC or EMS varied according to the treatment temperature. In MMC- or EMS-exposed cultures, the SCE frequency increased continuously with increasing treatment temperature; treatment at 37 degrees C resulted in a 3-4 times greater induction of SCEs than did that at room temperature (25 degrees C). On the other hand, SCE frequencies in cells exposed to 4NQO remained within normal deviation, showing no temperature-dependent changes. Baseline SCE frequencies remained almost constant within the temperature range tested. These data indicate that treatment temperature is a very critical factor in determining the sensitivity of cells to the chemical induction of SCEs.     

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.     

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.     

Studies on the utilization of a plant SCE test in detecting potential mutagenic agents

In this paper a modified procedure for sister-chromatid differentiation in plant cells is reported. Using this procedure some chemicals were tested for SCE induction in Vicia faba, Hordeum vulgare and Secale cereale. The chemicals tested were ethanol, chromium oxide, sodium saccharin, fluorouracil, ascorbic acid (vitamin c), omethoate and phenol. The experimental results showed that most of them induced SCE increases in mouse spleen cells, human lymphocytes and plant cells. The increase of SCEs per cell in plant cells is in agreement with that found in human lymphocytes or in mouse spleen cells. In our opinion, the utilization of SCE in plants is a simple and inexpensive technique for detecting potential mutagenic agents in the environment.     

The effect of a tumor promotor, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), on sister-chromatid exchange formation in cultured Chinese hamster cells.

The tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) does not increase the sister-chromatid exchange (SCE) frequency in either Chinese hamster ovary (CHO) or lung (V 79) cells which are cultured in the presence of 5-bromodeoxyuridine. Moreover, TPA does not alter the induction of SCEs in CHO cells by mitomycin C during the first 3 cycles following addition of the alkylating agent. These SCE induction data do not by themselves support the hypothesis that tumor promotion by TPA depends on the enhancement of mitotic recombination.     

Suppressing effect of tannic acid on the frequencies of mutagen-induced sister-chromatid exchanges in mammalian cells

The effects of tannic acid (m-galloyl gallic acid) and 7 of its analogues on the frequencies of sister-chromatid exchanges (SCEs) were investigated in cultured Chinese hamster cells. SCEs induced by UV-light or mitomycin C (MMC) were suppressed by post-treatment with tannic acid and 5 of its analogues. These effects were independent of the extension of the cell cycle. The compounds which showed an SCE-suppressing effect have a common structure of 3 neighboring hydroxy or methoxy groups substituted on the phenyl group in benzoic acid or ester. These decreasing effects of tannic acid were observed in the G1 phase but not in the S or G2 phase of the cell cycle and a greater decline of the frequencies of UV-induced SCEs during liquid holding was seen in the presence of tannic acid. However, cells irradiated with X-rays were not influenced by tannic acid. In cells from a xeroderma pigmentosum (XP) patient, a Fanconi's anemia (FA) patient, and a normal human embryo, MMC-induced SCEs were also decreased by post-treatment with tannic acid. Tannic acid reduced the SCE frequencies in UV-irradiated FA and normal human cells but not in UV-irradiated XP cells. Our results suggest that tannic acid modifies DNA-excision repair and that the decrease in the amount of unrepaired DNA damage might cause the reduction of induced SCEs.     

The effect of glutathione depletion on sister-chromatid exchange induction by cytostatic drugs

Using sister-chromatid exchanges (SCEs) as an indicator for DNA damage, we investigated the role of glutathione (GSH) as a determinant of cellular sensitivity to the DNA-damaging effects of the cytostatic drugs adriamycin (AM) and cyclophosphamide (CP). Exposure of V79 cells to buthionine sulfoximine (BSO) resulted in a complete depletion of cellular GSH content without toxicity and without increasing the SCE frequency. Subsequent 3-h treatment of GSH-depleted cells with AM or S9-mix-activated CP caused a potentiation of SCE induction. In Chinese hamster ovary (CHO) cells, which showed a higher GSH level compared to V79 cells, BSO treatment led to a depletion of GSH to about 5% of the control and increased SCE induction by AM and CP. Compared to V79 cells, the effect of AM on SCE frequencies was less distinct in CHO cells, while CP exerted a similar effect in both cell lines. Pretreatment of V79 cells with GSH increased the cellular GSH content, but had no effect on the induction of SCEs by AM, and pretreatment with cysteine influenced neither GSH levels nor SCE induction by AM. The study shows that SCEs are a suitable indicator for testing the modulation of of drug genotoxicity by GSH. The importance of different GSH contents of cell lines for their response to mutagens is discussed.     

Are SCE frequencies indicative of adaptive response of plant cells?

Low-dose pretreatments with maleic hydrazide, mitomycin C, and N-methyl-N-nitrosourea or sublethal heat shock were tested with regard to their effect on sister-chromatid exchange (SCE) induction by high doses of the same mutagens administered 2 h later to root-tip meristems of Vicia faba. Consecutive treatments resulted in either additive or, in a minority of experiments, in below-additive SCE frequencies. A model is proposed to explain the conflicting data reported on adaptation to SCE and aberration induction.     

Different mutations responsible for the elevated sister-chromatid exchange frequencies in Bloom syndrome and X-irradiated B-lymphoblastoid cell lines originating from acute leukemia

Cell hybridization and co-cultivation protocols have been used to determine whether the increased rates of sister-chromatid exchanges (SCEs) exhibited by Bloom syndrome (BS) and a human mutant cell line (CCRF-SB-T1), originating from an X-irradiated acute leukemia-derived B-lymphoblastoid cell line, have the same or different bases. Cell fusion of CCRF-SB-T1 with each of 4 different BS B-lymphoblastoid cell lines (LCLs), retaining a high-SCE character, exhibited low (normal level) numbers of SCEs, signifying complementation. Co-cultivation of CCRF-SB-T1 and BS B-LCLs also resulted in a significant reduction in SCE level, from 70 to 35, in BS cells, lowered the BrdU concentrations necessary for sister-chromatid differential staining (SCD) from 15 micrograms/ml (0.05 mM) to 2.0 micrograms/ml (0.01 mM) and resulted in a completely normal level of SCE in CCRF-SB-T1 cells. This strongly suggests that the defects in the 2 cell types are different. In the assay of cell extracts, the 4 BS cell lines appear to have lost thymidylate (TMP) synthetase activity (about 50% reduction from that of normal cells), whereas CCRF-SB-T1 cells show a 20% increase of TMP synthetase activity compared to normal cells.     

Roles of DNA interstrand crosslinking and its repair in the induction of sister-chromatid exchange and a higher induction in fanconi's anemia cells

The roles of DNA crosslink and its repair in the induction of sister-chromatid exchanges (SCEs) were studied in normal, xeroderma pigmentosum (XP) complementation group A, and Fanconi's anemia (FA) fibroblasts after treatment with mitomycin C (MC) or decarbamoyl mitomycin C (DMC) for 1 h. FA strains were 5—30-fold more sensitive to MC killing than normal cells, but normally responded to DMC killing. XP group-A cells were twice and only slightly more sensitive to DMC and MC killings, respectively, than normal cells. The induction rate of immediate SCEs by MC was 1.7 times higher, despite a normal SCE rate by DMC, in FA strains than that in normal cells. Alternatively, SCE rates by DMC and MC were 6 times and only 1.3 times higher, respectively, in XP cells than in normal cells. In normal cells, the reduction of MC-induced SCEs as a function of repair time followed a biphasic curve of the first rapid (half-life, 2 h) and the second slow (half-life, 14 h) components. Such components corresponded exactly to the first half-excision and the second slow repair processes of molecular crosslink repair. In MC-induced SCEs, FA17JTO cells exhibited only the slow reduction component without the first rapid component and a higher saturation level in the time-dependent reduction in SCEs. This indicates that SCEs are produced by crosslinks remaining unrepaired for long times (24—48 h) after treatment of FA cells. Conversely, XP group-A cells capable of the first half-excision manifested the first rapid reduction in SCEs, although the second component declined at the slowest rate (half-life, 48 h) owing to a defect in the second mono-adduct repair. The reduction in DMC-induced SCEs followed only the slow component. Thus, these results demonstrate that crosslink can be the lesion leading to SCE, and the MC-induced SCE frequency is higher in FA cells than in normal cells. In the FA20JTO strain, such a repair defect seemed to be less than in FA17JTO cells, judged from the survival and SCE characteristics.     

Ultraviolet light and mitomycin C induced sister-chromatid exchanges in fibroblasts from patients with retinoblastoma

Ultraviolet light and mitomycin C (MMC) induced sister-chromatid exchanges (SCEs) were investigated in 6 diploid fibroblast strains derived from 3 patients with deletion 13 and retinoblastoma, one patient with a hereditary form of retinoblastoma, one patient with trisomy 13, and one normal control. Two fibroblast strains with del(13)(q14q22) showed a significant increase in SCEs compared to the control after UV and MMC treatments. In contrast, cell strains with del(13)(q12q14) and trisomy 13 did not show increased SCEs. The frequency of SCEs in fibroblasts from a patient with autosomal dominant retinoblastomas (no deletions) was significantly increased by UV, but not by MMC. The results suggest that cell strains with different deletions of chromosome 13 have different SCE responses to UV and MMC inductions. The cells with del(13)(q14q22) may have a DNA-repair defect.     

Increased sister-chromatid exchanges (SCEs) and chromosomal fragilities by BrdU in a human mutant B-lymphoblastoid cell line

From an X-irradiated human B-lymphoblastoid cell line (CCRF-SB), we have isolated a unique mutant clone (CCRF-SB-T1) which reveals high frequencies of sister-chromatid exchanges (SCEs) and chromosomal fragilities in the C-band regions of chromosomes Nos. 1, 9 and 16, when exposed to high concentrations of bromodeoxyuridine (BrdU). A clear BrdU dose-dependent increase of SCEs (9.6 SCEs/cell at 0.05 mM, 40 SCEs/cell at 0.37 mM on average) in this mutant was observed. Relative contributions of nucleoside and a thymidine (dT) analog of BrdU to high SCEs were studied, since an unusual SCE response to BrdU led us to suspect the significance of BrdU incorporation into DNA and dT pool disturbances. Addition of deoxycytidine (dC), dT or both dC and dT causes an increase of SCEs. On the other hand, deoxyadenosine (dA) and deoxyguanosine (dG) did not have significant effects on SCEs in SB-T1 cells. These results suggest that disturbances of pyrimidine-nucleotide synthesis, including gross imbalance of nucleotide pools, play a pivotal role in the high SCE induction of SB-T1 cells by BrdU.     

Cell-stage dependence of mutagen-induced sister-chromatid exchanges in human lymphocyte cultures

The induction of sister-chromatid exchanges (SCEs) was studied in phytohemagglutinin (PHA)-stimulated human lymphocytes exposed for 1 h to mitomycin C (MMC, 3 X 10(-6) M), ethyl methanesulphonate (EMS, 2 X 10(-2) M), or 4-nitroquinoline-1-oxide (4NQO, 3 X 10(-5) M) at various cell-cycle stages of 72-h cultures. The doses of the chemical were chosen to give about 20 SCEs per cell when treated at Go. The SCE frequency increased almost linearly with MMC or EMS treatments at later times after PHA stimulation, peaking with those at 36 h (at around the first G1/S boundary in the 2 consecutive cell cycles, which was revealed by concomitant experiments), and then decreased with subsequent treatment times. Cell-cycle kinetics and the cell stages at which the cells were treated were measured by autoradiography and sister-chromatid differential staining. The data show that MMC and EMS produce larger numbers of SCEs when treated at stages closer to the beginning of S, and that the most efficient time of treatment is the G1/S boundary in the first cell cycle of the two consecutive cycles before sampling. Pulse treatment with EMS caused about 3 times larger inductions of SCEs when done at late G1/early S(G1/S boundary) in the first cell cycle compared to that at G0/early G1, whereas identical exposure to MMC at the first G1/S boundary produced only 1.5 times larger numbers of SCEs than that at G0/early G1. EMS and MMC both, however, induced 30-40% larger numbers of SCEs when treated at the G1/S boundary in the first cell cycle than when treated at the second cell cycle before sampling. On the contrary, treatment with 4NQO led to the induction of about the same numbers of SCEs even when treated at different cell-cycle stages before the second G1/S boundary. The SCE frequency in 4NQO-treated cells then decreased with subsequent treatment times.     

The influence of incorporated bromodeoxyuridine on mutagenicity testing by sister chromatid exchange induction inVicia faba root tip cells

The induction of sister chromatid exchanges (SCEs) inVicia faba root-tip cells after short-term (2 h) and long-term (24 h) treatments with alkylating agents (N-methyl-N-nitrosourea, ethyl methanesulphonate) and maleic hydrazide was studied. The primary roots were treated with mutagens before or after 5-bromodeoxyuridine (BrdU) incorporation into DNA and the influence of mutagen application on SCE induction in the cells with non- and BrdU-substituted chromosomal DNA. On the contrary, application of maleic hydrazide after the incorporation of BrdU into DNA strongly increased the rate of SCEs. The lowest limit concentrations of mutagens capable of significantly increasing SCE frequency in the cells with non-substituted DNA after the long-term treatment were estimated.     

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.     

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

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

Antagonizing effect of 3-aminoharman on induction of sister-chromatid exchanges by mutagens

3-Aminoharman (3AH, 3-amino-1-methyl-9H-pyrido[3,4-b]indole), which has been reported as a novel substance with an antagonistic effect on induction of sister-chromatid exchange (SCE) by polycyclic mutagens in the presence of the metabolic activation system, was examined with a cultured human lymphoblastoid cell line, NL3, for its effect on SCE induction by direct-acting mutagens such as mitomycin C (MMC), nitrogen mustard N-oxide (NMO), methyl methanesulfonate (MMS), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitroquinoline 1-oxide (4NQO) and 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (OH-Trp-P-2), and also by ultraviolet light (UV) irradiation. The results obtained on simultaneous treatment with 3AH and mutagens were as follows: (1) 3AH suppressed more than 50% of SCEs induced by MMC, NMO and OH-Trp-P-2; (2) 4NQO- and MNNG-induced SCEs were also suppressed by 3AH but to a lesser degree; (3) MMS-induced SCEs were not, however, altered by 3AH; and (4) the suppression of SCE by 3AH was dose-dependent. Treatment of cells with 3AH for 2 h immediately before MMC exposure suppressed SCE induction to a significant degree similar to the simultaneous treatment, but post-treatment with 3AH was much less effective. 3AH inhibited SCE induction by NMO when 3AH treatment was carried out either before or after NMO treatment, to an extent similar to the simultaneous treatment. Treatments with 3AH either before or after UV exposure did not change the UV-induced SCEs. Results with these direct-acting mutagens ruled out the relevance of metabolic activation as a necessary step for the antagonizing effect of 3AH.