Distribution of sister chromatid exchanges in chromosomes of normal Chinese hamster and its cell lines exposed to BrdU and MMC

Sister chromatid exchanges (SCEs) were investigated in chromosomes from normal male Chinese hamster (CH) and its cell lines (CHW, 1102 and 1103). The fibroblasts were grown for two replication cycles in medium containing BrdU and mitomycin C (MMC) at concentrations of 0.01, 0.02 and 0.03 micrograms/ml of medium. The difference in SCEs/cell between male CH and CHW was negligible, but the difference between CHW and 1102 was about 2.6-fold. It is suggested from karyotypic differences between CHW and 1102, that the control of SCEs might be due partly or completely to chromosome 5 in Chinese hamster. The lines CHW and 1102 were less responsive than normal Chinese hamster cells when exposed to different MMC concentrations. It is suggested that the lines CHW and 1102 might be slightly resistant to MMC. The frequency of SCEs decreased with the decrease of chromosome size. SCEs are not preferentially distributed on any autosomal chromosomes. No SCEs were found in normal X-chromosomes. The majority of exchanges appear to be either interband regions or very near band-interband junctions.     

Antagonization by cycloheximide of ethyl methanesulfonate-induced 6-thioguanine-resistant mutation and sister-chromatid exchanges

We studied the effect of cycloheximide (CH) on the induction of mutation and sister-chromatid exchanges (SCEs) in ethyl methanesulfonate (EMS)-treated Chinese hamster cells. At 10^?6 M, CH strongly antagonized the induction of mutation and SCEs and cell survival increased. This suggests that protein synthesis is essential for the induction of mutation as well as SCEs. Results of experiments in which CH treatment preceded or followed exposure to mutagens were similar with respect to the response curves obtained for mutation and SCEs.     

Synergistic action of cysteamine and BrdU-substituted DNA in the induction of sister chromatid exchanges

The effect of different BrdU-concentrations on the cysteamine-induced SCE-rate was investigated in V-79 Chinese hamster cell monolayer cultures. Both cysteamine and its auto-oxidation product cystamine act synergistically with BrdU in the induction of SCEs. A given concentration of these substances produces a low SCE-frequency at low BrdU-concentrations — but the incidence of SCEs is significantly increased at increased BrdU-concentrations. — Using one-cycle substitution experiments and the determination of the relative level of substitution by means of ³HBrdU-incorporation, this synergism was shown to depend on the BrdU incorporated in the DNA and on the extent to which this incorporation takes place.     

The effect of sulfhydryl compounds on sister-chromatid exchanges: II. The question of cell specificity and the role of H2O2

In contrast with earlier report on the induction of sister-chromatid exchanges (SCEs) by SH compounds in cell lines of the Chinese hamster, cysteine, cysteamine and cystamine did not cause an increase of the SCE frequency in human lymphocyte cultures. Differences in the treatment protocols or variations of the Brd Urd concentration had no effect on the induction of SCEs by these substances. The inclusion of H₂O₂ and comparative investigations with V79 cells of the Chinese hamster showed that the probable reason for the SCE induction by SH compounds is the inability of the cells to degrade H₂O₂.Furthermore, for cystamine it became clear that additional effects must exist besides the induction of SCEs through H₂O₂.The present study underlines the fact that the examination of a substance within one cell system does not necessarily permit a reliable statement about the DNA-damaging property of this substance.     

Effect of novobiocin on the frequencies of chromatid-type aberrations and sister-chromatid exchanges following gamma-irradiation

The effect of novobiocin on the frequencies of chromatid-type aberrations and SCEs was examined in Chinese hamster V79 cells which were exposed to gamma-rays and post-treated with novobiocin. While no chromatid aberrations were induced in the unirradiated cells by novobiocin, the frequency of SCEs was slightly increased by treatment with novobiocin alone. Irradiation of G2 cells produced multiple chromatid-type aberrations and post-treatment of the irradiated cells with novobiocin resulted in a significant increase of the aberrations, including chromatid gaps and breaks. In contrast, novobiocin failed to increase the frequency of SCEs induced by gamma-rays when the irradiated cells were post-treated with novobiocin.     

Enhancing effects of cinoxate and methyl sinapate on the frequencies of sister-chromatid exchanges and chromosome aberrations in cultured mammalian cells

Sister-chromatid exchanges (SCEs) induced by mitomycin C (MMC), 4-nitroquinoline-1-oxide (4NQO) or UV-light in cultured Chinese hamster ovary cells (CHO K-1 cells) were enhanced by cinoxate (2-ethoxyethyl p-methoxycinnamate) or methyl sinapate (methyl 3,5-dimethoxy 4-hydroxycinnamate). Both substances are cinnamate derivatives and cinoxate is commonly used as a cosmetic UV absorber. Methyl sinapate also increased the frequency of cells with chromosome aberrations in the CHO K-1 cells treated with MMC, 4NQO or UV. These increasing effects of methyl sinapate were critical in the G1 phase of the cell cycle and the decline of the frequencies of UV-induced SCEs and chromosome aberrations during liquid holding was not seen in the presence of methyl sinapate. Both compounds were, however, ineffective in cells treated with X-rays. In cells from a normal human embryo and from a xeroderma pigmentosum (XP) patient, MMC-induced SCEs were also increased by the post-treatment with methyl sinapate. The SCE frequencies in UV-irradiated normal human cells were elevated by methyl sinapate, but no SCE-enhancing effects were observed in UV-irradiated XP cells. Our results suggest that the test substances inhibit DNA excision repair and that the increase in the amount of unrepaired DNA damage might cause the enhancement of induced SCEs and chromosome aberrations.     

Effects of vanillin on sister-chromatid exchanges and chromosome aberrations induced by mitomycin C in cultured Chinese hamster ovary cells

Effects of vanillin on the induction of sister-chromatid exchanges (SCEs) and structural chromosome aberrations by mitomycin C (MMC) were investigated in cultured Chinese hamster ovary cells. Vanillin induced neither SCEs nor chromosome aberrations by itself. However, an obvious increase in the frequency of SCEs was observed when MMC-treated cells were cultured in the presence of vanillin. The effect of vanillin was S-phase-dependent. On the contrary, the frequency of cells with chromosome aberrations was significantly decreased by the post-treatment with vanillin at G2 phase.     

Uncoupling of the induction of mutations and sister-chromatid exchanges by the replication of 5-bromouracil-substituted DNA

The REP mutagenesis protocol, which involves the replication of 5-bromouracil (BrUra)-substituted DNA in the presence of deoxyribonucleoside triphosphate (dNTP) pool imbalance, has been shown to induce both mutations and sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. However, when a Syrian hamster melanoma-derived cell line, called 2E, which was selected for its ability to replace all of the thymine residues in DNA with BrUra, was subjected to the REP mutagenesis protocol, the correlation between the induction of mutations and SCEs was no longer observed. The 2E cells were found to be much more sensitive to the induction of mutations by REP mutagenesis than were the CHO cells. This increased sensitivity to REP mutagenesis was found to correlate with increased perturbations of the dNTP pools that have been shown to be involved in the mutagenic mechanism of this protocol. In contrast, when the induction of SCEs by the REP protocol was measured, it was found that although a baseline level of SCEs was detected in 2E cells, no significant induction of SCEs due to dNTP pool perturbation was observed. It was shown that high levels of SCEs were readily induced in 2E cells by other agents, e.g. mitomycin C. A model, which discusses the fate of mismatched bases thought to be generated by the REP mutagenesis protocol as the determining factor for the induction of mutations of SCEs, is proposed to explain the uncoupling of mutagenesis and SCE induction in 2E cells.     

Yield of SCEs and translocations produced by 3 aminobenzamide in cultured Chinese hamster cells

Different concentrations of 3-aminobenzamide (3AB), a strong inhibitor of poly(ADP-ribose) polymerase (PARP), were used to study their effect on the BrdU-substituted DNA of the Chinese hamster AA8 cell line. The frequencies of sister chromatid exchanges (SCEs) and translocations were determined using the fluorescence plus Giemsa (FPG) and fluorescence in situ hybridization (FISH) techniques, respectively. The results indicate that 3AB effectively induced a dose-dependent increase in the frequency of SCEs, but this enhancement in the yield of SCEs was not paralleled by an increase in translocations. These results are discussed in terms of the as yet poorly understood molecular mechanisms of action of the enzyme PARP.     

SCE induction and cell-cycle delay by toxaphene

Toxaphene is genotoxic in mammalian cell systems and also inhibits cell replication. It was therefore used to investigate possible masking of SCE induction due to cell-cycle delay. In this study, toxaphene-treated Chinese hamster lung (Don) cells exhibited a dose-dependent decrease in cell-cycle progression compared with untreated cells. At high, nontoxic toxaphene levels (15 micrograms/ml), cell cycling also slowed as the toxaphene treatment time was increased. Toxaphene induced significantly higher numbers of SCEs in treated cells, demonstrating a dose- and treatment time-relationship. Slopes of dose-response curves were 0.29, 0.43 and 0.77 SCE/micrograms toxaphene for 20.5 h, 24.5 h and 28.5 h incubation, respectively. There were no changes in SCE values in control cultures even when slower dividing cells were sampled e.g. at longer incubation times. Thus, higher SCE values in Chinese hamster cells were not associated per se with slower or more delayed cells. The results demonstrate that longer toxaphene treatment times were not necessary for obtaining sufficient harlequin-stained cells for SCE analysis, but that higher numbers of SCEs occurred in slower dividing cells, following prolonged incubation of cultures treated with toxaphene.     

Plant extracts induce chromosome aberrations and sister-chromatid exchanges in Chinese hamster ovary cells and human lymphocytes

Effects of extracts from Vicia faba were compared with those of Zea mays for the induction of sister-chromatid exchanges (SCEs) and of chromosome aberrations (CAs) in Chinese hamster ovary (CHO) cells. CA induction by the maize extract was also tested in human lymphocytes. The extracts from roots and leaves of Vicia faba induced CAs and SCEs in CHO cells. The extracts from maize leaves also induced SCEs and CAs in CHO cells, and CAs in human lymphocytes. Maize extracts were more potent in inducing SCEs than Vicia extracts and the SCE- and CA-inducing capacity of maize extracts decreased during preincubation before addition to cells.     

Effect of cigarette-smoke condensate and norharman on the induction of SCEs by direct and indirect mutagens in CHO cells

Cigarette-smoke condensate and norharman were investigated either alone or in combination with a number of direct or indirect mutagens for the induction of sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. Cigarette-smoke condensate and norharman induced SCEs in these cells, only in the presence of a metabolic activation system. The number of SCEs induced by the direct-acting mutagens mitomycin C and N-methyl-N'-nitro-N-nitrosoguanidine was decreased in the presence of cigarette-smoke condensate or norharman. However, cigarette-smoke condensate and norharman showed synergistic effects in combination with the indirect mutagens 2-acetylaminofluorene, 2-aminofluorene, N-hydroxy-acetylaminofluorene, 2-aminoanthracene and benzo[a]pyrene. No synergism was observed when CHO cells were treated simultaneously with cigarette-smoke condensate or norharman and the indirect mutagen cyclophosphamide.     

Contribution of incorporated 5-bromodeoxyuridine in DNA to the frequencies of sister-chromatid exchanges induced by inhibitors of poly-(ADP-ribose)-polymerase

3-Aminobenzamide and benzamide, two potent inhibitors of poly-(ADP-ribose)-polymerase increase the frequencies of SCEs in Chinese hamster ovary cells in a dose-dependent manner. SCEs were studied in cells in which the inhibitors were present either during the first cell cycle or the second cell cycle or both. Most of the induced SCEs were found to be formed during the second cell cycle in which BU-containing DNA was used as template for DNA synthesis. In cells which were pregrown for 4 cell cycles in the presence of BrdUrd, in order to obtain both sister chromatids bifiliarly substituted with BU in their DNA, it was found that the presence of inhibitor even in the first cell cycle increased the frequencies of SCEs. It is concluded that the incorporated BrdUrd plays an important role in the origin of spontaneous and induced SCEs. 3-Aminobenzamide alone or benzamide in the presence of BrdUrd during culture, did not increase the frequencies of mutations to HGPRT^? in these cells.     

Detection of bromodeoxyuridine incorporation in mammalian chromosomes by a bromodeoxyuridine antibody

A commercially available bromodeoxyuridine (BrdUrd) antibody was used to demonstrate sister chromatid differentiation (SCD) and to evaluate sister chromatid exchanges (SCEs) in V79 Chinese hamster cells. V79 cells were cultivated for one cell cycle in the presence of BrdUrd, followed by a second cell cycle in the absence of BrdUrd. Chromosome preparations were stained by a common immunologic staining technique. The staining pattern observed is similar to that after FPG (fluorescent plus Giemsa) staining, though with reverse staining specificity. The sensitivity of BrdUrd detection is enhanced by a factor of 20 compared to the FPG technique and thus allows the evaluation of SCEs at very low BrdUrd concentrations. The application of the antibody technique gives information about the origin and localization of SCEs and produces further evidence for the spontaneous occurrence of SCEs.     

Characterization of sister chromatid exchange induction by 8-methoxypsoralen plus near UV light

The combination of 8-methoxypsoralen and near UV light is highly effective in inducing sister chromatid exchanges (SCEs) in Chinese hamster ovary cells. Appreciable increases in SCEs can be effected by treatments compatible with cell survival, and effects of a single dose of alkylation persist over multiple generations. Both the frequency and location of SCEs induced at different times within the DNA synthesis period varies in a manner indicating that exchange induction is restricted to regions which replicated during or after DNA damage.     

Effects of antimutagenic flavourings on SCEs induced by chemical mutagens in cultured Chinese hamster cells

Effects of antimutagenic flavourings such as vanillin, ethylvanillin, anisaldehyde, cinnamaldehyde, coumarin and umbelliferone on the induction of SCEs by MMC were investigated in cultured Chinese hamster ovary cells. None of these 6 flavourings showed any SCE-inducing activity by themselves. However, an obvious increase in the frequencies of SCEs was observed when MMC-pretreated cells were cultured in the presence of each flavouring. All these compounds have either an alpha, beta-unsaturated carbonyl group or a carbonyl functionality neighbouring the phenyl group which may react with an enzyme SH-group and cause higher-order structure changes. SCE-enhancing effects of vanillin were further investigated on 6 other kinds of mutagens. Vanillin was also effective on SCEs induced by EMS, ENNG, ENU or MNU. On the other hand, MMS- or MNNG-induced SCEs were not influenced at all by vanillin. SCE-enhancing effects of vanillin seemed to be dependent on the quality of lesions in DNA.     

Effects of a tumor promoter and an anti-promoter on spontaneous and UV-induced 6-thioguanine-resistant mutations and sister-chromatid exchanges in V79 Chinese hamster cells

The effects of a tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or an anti-promotor antipain (protease inhibitor) on spontaneous and ultraviolet-induced sister-chromatid exchanges (SCEs) and 6-thioguanine- resistant (6TG^r) recessive mutations were examined in V79 Chinese hamster cells in culture. TPA and/or antipain neither significantly altered base-line and UV-induced immediate SCE frequencies, nor decreased the level of delayed SCEs which persisted 6–7 days after irradiation. TPA and/or antipain appeared to enhance the recovery of UV-induced 6TG^r colonies at the plateau expression phase despite non-mutagenicity by themselves and unaltered metabolic co- operation. Thus, the results conceivably imply that the 6TG^r-recessive mutation expression, but not fixation, can be modulated at the cell level by the TPA and/or antipain. Our results, together with the recent results of Loveday and Latt, may argue against the notion that TPA enhances the antipain-suppressible SCEs as an index of mitotic recombination in relevance with a tumor-promotion mechanism.     

Induction of sister-chromatid exchanges by pesticides in primary rat tracheal epithelial cells and Chinese hamster ovary cells

Possible induction of sister-chromatid exchanges by butachlor, paraquat, phorate and monocrotophos was examined in primary rat tracheal epithelial (RTE) and Chinese hamster ovary (CHO) cells. At dose levels that killed less than 50% of the cell population, monocrotophos induced SCEs positively in CHO and RTE cells, while paraquat was positive only in RTE cells. In two trials of the same experiment, paraquat and butachlor in CHO cells, and phorate in either RTE or CHO cells failed to induce a significant number of SCEs at any dose level within the ranges assayed. On the other hand, in RTE cells, butachlor induced a significant number of SCEs at a dose level of 5 micrograms/ml in one trial, but was insignificant in another. The inductions in these assays were, however, dose-dependent. The addition of S9 mixture did not alter the results of SCE induction by these 4 pesticides in CHO cells. RTE cells were more vulnerable to paraquat in cytotoxicity and SCE assays than CHO cells. Cytotoxicities were ranked as butachlor greater than phorate greater than paraquat greater than monocrotophos to CHO cells and paraquat greater than butachlor greater than phorate greater than monocrotophos to RTE cells. Significant cell cycle delays were only found in the treatments with the highest dose levels of butachlor, paraquat and phorate in CHO cells. In addition, this is the first report on SCE induction in RTE cells.     

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.     

The effect of 1,10-phenanthroline on the chromosome damage and sister-chromatid exchanges induced by streptozotocin in mammalian and insect cells

The effect of the metal chelating agent 1,10-Phenanthroline (PNT) on the streptozotocin (STZ)-induced chromosomal aberrations (CAs) and sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) and mosquito (Aedes albopictus) cells was investigated. Treatment of CHO and mosquito cells with STZ produced a significant and dose-response increase in the yield of CAs as well as SCEs (p<0.05). The addition of PNT prevented the induction of CAs by STZ in both types of cells, causing a significant decrease in the frequency of STZ-induced CAs (46.5-72.5%) (p<0.05). This fact indicates that intracellular transition metals are implicated in STZ-induced CAs and that the Fenton reaction (Fe(2+)+H(2)O(2)-->OH degrees +OH(-)+ Fe(3+)) is partly responsible for the production of CAs by this compound. On the other hand, the addition of PNT to CHO and mosquito cell cultures did not prevent the induction of SCEs by STZ. Therefore, it is valid to assume that the induction of CAs and SCEs by STZ occurs by different mechanisms.