SCE induction in Chinese hamster ovary cells (CHO) exposed to G agents

Cultured Chinese hamster ovary (CHO) cells were exposed to two neurotoxic organophosphates, either satin (GBI, GBII) at 1.4 x 10⁻³ M or soman (GD) at 1.1 and 2.2 x 10^t-3 M for 1 h, grown and their metaphase chromosomes scored for sister-chromatid exchanges (SCE). No cytotoxicity was seen with either agent at any dose level tested. Since histograms of SCE per cell showed that they were non-symmetrically arrayed around the mean, the number of SCEs were analyzed by using the nonparametric tests, Mann-Whitney and Kruskall-Wallis. Agents GBI and GBII did not show any significant increase in SCE over baseline. On the other hand, GD demonstrated a statistically significant increase in SCE with and without metabolic activation. Ethyl methanesulfonate (EMS) alone at 5 x 10⁻³ M and cyclophosphamide (CP) at 10⁻⁴ M in the presence of rat microsomes (S9) induced a 3- and 8-fold increase in SCE per cell, respectively.     

Induction of sister chromatid exchanges in Chinese hamster cells by carcinogenic mutagens requiring metabolic activation

The induction of SCEs has proven to be the most sensitive mammalian system for detecting the effects of mutagenic carcinogens. Several chemicals that are mutagenic in the exquisitely sensitive Salmonella mutagenesis test have now been tested in Chinese hamster ovary (CHO) cells in culture. Cells were grown for 24 h (two rounds of DNA replication) in the presence of bromodeoxyuridine (Brd Urd) to form harlequin chromosomes in which it is possible to see the SCEs. To test whether the chemicals increase SCEs without metabolic activation, they were added at various concentrations for the entire culture period. To test if they induce SCEs after activation they were added for 30 min along with microsomes from rat liver (S-9 Mix of Ames). After this treatment the cells were cultured with Brd Urd. N-hydrosy-2-acetylamino-fluorene (10^?6?10^?4 M), N-acetoxy-2-acetylaminofluorenee (10)^?9?10^?7 M), and aflatoxin B₁ (10^?6?10^?4 M) all increased the yield of SCEs with increasing concentration. Further, aflatoxin B₁ was dramatically activated by the addition of rat liver microsomes. Benzo(a)pyrene (10^?6?10^?4 M), however, gave an increase only when activated. 2-aminofluorene (10^?6?10^?4 M) gave a slight increase only after long treatments without activation. In no case did 2-acetylamino-fluorene (10^?6?10^?4 M) increase SCE's. It thus appears that some of the chemicals that are positive in the Salmonella system are negative in the mammalian SCE system. Whether this reflects a difference in sensitivity between the two tests or the ability of the SCE test to discriminate between those chemicals that are active in bacteria, but not in mammals, is as yet unknown.     

Analysis of bromodeoxyuridine-induced single and twin sister chromatid exchanges in tetraploid Chinese hamster ovary cells

Culture of cells in high exogenous levels (>10^–4 M) of bromodeoxyuridine (BrdUrd) or thymidine will increase the baseline sister chromatid exchange (SCE) frequency. The effect is thought to be related to the balance of the DNA precursors thymidine and deoxycytidine. Exogenous addition of deoxycytidine will reverse this effect. Single and twin SCEs were analysed in Colcemid-induced tetraploid Chinese hamster ovary cells exposed to different concentrations of BrdUrd to determine at what stage SCEs are induced by high levels of BrdUrd. In cells exposed to low concentrations of BrdUrd (10^–5 M), equal numbers of SCEs were induced in each of the two cell cycles. With increasing concentrations of BrdUrd (10^–4 to 2×10^–4 M), SCE frequency increased in both cell cycles, but far more SCEs were induced in the second cell cycle. Deoxycytidine (2×10^–4 M) reduced the frequency of SCEs primarily by reducing the frequency of SCEs induced in the second cell cycle. Treatment with 3-aminobenzamide (3AB), a potent inhibitor of poly(ADP-ribose) polymerase, produced effects similar to exposure to high levels of BrdUrd including inducing SCEs in the second replication cycle. This suggests a similar mechanism of action. Deoxycytidine had no effect on 3AB-induced SCEs, however, and there was no interaction between 3AB and high exogenous levels of BrdUrd in SCE induction. Thus these two agents probably act through different mechanisms.     

Sister chromatid exchanges induced in cultured mammalian cells by chromate

Chromate compounds induced sister chromatoid exchanges (SCEs) and chromosome aberrations in cultured mammalian cells. Similar increases in SCE frequency were observed in human fibroblasts exposed to the compounds K2Cr2O7 and K2CrO4. Marked increases in SCE frequency in cells exposed to chromate for a 48-h period were detected at concentrations between 10(-7) and 10(-6) M. Chromosome aberrations (primarily chromatid breaks) were also produced in human cells exposed to K2CrO4 at concentrations between 8 . 10(-7) and 3 . 10(-6) M. K2CrO4, but not the trivalent compound CrCl3, induced SCEs in Chinese hamster ovary (CHO) cells at low concentrations.     

Effects of temperature on sister chromatid exchanges

Summary The influence of temperature on sister chromatid exchanges was investigated, and the results are discussed in connection with factors possibly involved in temperature-induced SCE-formation.Whereas the SCE frequency increased with increasing growth temperature in a cell line of Xenopus laevis (EAX), which permits the examination of great temperature differences, a Chinese hamster cell line (V-79) revealed a U-shaped temperature-response curve. In addition, it was found that cold treatment at 4°C caused an induction of SCEs in the V-79 cell line.Different BrdU concentrations had no effect on the temperature-induced SCE frequencies and mitomycin C led to an induction of SCEs parallel to the base-line values at different temperatures.     

Evaluation of the mutagenic potential of the hair dye N-methyl-amino-2-nitro-4-N′,N′-bis-(2-hydroxyethyl)-aminobenzene in a battery consisting of Drosophila and mammalian cytogenetic assays

The compound N-methyl-amino-2-nitro-4-N′,N′-bis-(2-hydroxyethyl)-aminobenzene was tested for mutagenic activity in the sex-linked recessive lethal test with Drosophila melanogaster, the induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) with Chinese hamster ovary (CHO) cells in vitro, and the micronucleus test with mouse bone-marrow cells in vivo. Consistently negative results were obtained with the 3 tests. The SCE tests gave positive results with prolonged treatments. It is concluded that reliable decisions about mutagenic activity cannot be based on the induction, in vitro, of SCEs alone.     

Sister chromatid induction by β-irradiation from incorporated 3H-thymidine: a paradox explained

Sister chromatid exchanges (SCE's) induced by [3H]thymidine (³HdT) of increasing specific activities incorporated over one cycle and 5-bromodeoxyuridine (BrdUrd) over the two following cycles were investigated in synchronised Chinese hamster ovary (CHO) cells. SCEs induced during the first cycle on a T.T template (SCE 1) show little increase with dose compared with those induced in the second cycle on a ³HT.T template (SCE 2) where the linear increase with dose reflects that seen after X irradiation. During the third cycle, SCEs 3.1 and 3.2 are induced on unlabelled T.B or labelled ³HT.B templates respectively. These templates are theoretically present in a 11 ratio after random segregation at second metaphase. Over practically the entire dose range however, the ratio 3.1/3.2, which dereased with dose, was >1.0 and similar to the high values obtained by other workers. At increasing times after BrdUrd introduction, the ratio decreased from >1.0 to <1.0. Measurements showed that the expected 50% level of labelled chromosomes at metaphase in the samples could vary between 42%–59%. Cells with >50% labelled chromosomes were more delayed in the cell cycle due to the ³H-irradiation than those with <50%. Early fixations therefore favoured SCE 3.1 while late favoured SCE 3.2. SCEs due to BrdUrd in ³HT.B and T.B templates showed no synergistic interaction with irradiation-induced SCEs. When these BrdUrd-induced SCEs were removed from the totals then the ³H-induced SCE levels in ³HT.T, and ³HT.B templates (SCE 2 and 3.2) were similar and increased at a similar rate with dose. This was 2–3 times faster than in SCE 1 and 3.1 where the SCE levels due to irradiation were again similar but lower than for 2 and 3.2. The -irradiation source is therefore most effective in inducing SCEs when present in the replicating fork and considerably less effective when it is just behind the fork (SCE 1) and/or in the surrounding chromosomes in the cell.     

Lack of induction of single-strand breaks in mammalian cells by sodium azide and its proximal mutagen

The mutagenicity of sodium azide in both higher plants and bacteria is well documented. However, in mammalian cells, research on the effects of azide on gene mutations has produced conflicting results. Furthermore, no research has been conducted on the effects of azide and its proximal mutagen (mutagenic metabolite) on DNA single-strand breaks. Experiments were designed to overcome this lack of information on azide mutagenicity and to evaluate the potential hazard of azide exposure to man.Chinese hamster V79 cells were treated with either azide or its proximal mutagen(s) for 2 or 6 h, respectively, and analyzed by alkaline elution for single-strand breaks. The data showed that neither azide nor the proximal mutagen(s) induced single-strand DNA breaks or DNA-protein cross-links.Therefore it appears that neither azide nor its proximal mutagen(s) interact directly with DNA and this suggests that azide may be an indirect-acting mutagen. Furthermore, this lack of interaction with DNA may account for azide's lack of carcinogenicity.     

Mutagenicity of nitrofuran derivatives, including furylfuramide, a food preservative.

The effects of sodium azide (NaN₃) in combination with diethyl sulfate (dES) or N-methyl-N′-nitrosourea (MNH) on mutation frequency in barley were studied. It was found that sodium azide produced high frequencies of chlorophyll mutations when used alone and has a synergistic effect on mutation yields following MNH treatments. However, the mutation frequency was decreased whe azide was applied following dES treatment of seeds. The mutagenic efficiency of azide was found to be high, possibly because of low “physiological” damage. The synergistic increase in mutation yields by MNH and azide treatment indicates that azide has unusual promise as a mutagen for both practical and research applications.     

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     

Sodium azide mutagenesis in mammals: inability of mammalian cells to convert azide to a mutagenic intermediate

Sodium azide is unique among mutagens. It is highly mutagenic in many plant and bacterial species but marginally mutagenic in mammalian cells. A possible explanation for this difference in mutagenic efficiency may lie in the inability of mammalian cells to convert azide to the putative ultimate mutagen. Normal human fibroblasts and Chinese hamster cells or cell-free extracts from these cell lines were treated with azide and the sonicates tested for mutagenicity in Salmonella strain TA1530. The data suggest that neither cell line was capable of converting azide to a mutagenic intermediate. In addition, both cell lines expressed the enzyme O-acetylserine(thio)-lyase which is responsible for the conversion of azide to azidoalanine, the putative mutagenic intermediate. Although mammalian cells possess the enzyme responsible for the conversion of azide to azidoalanine, they appear incapable of converting azide into a mutagenic intermediate in appreciable quantities. Further, the data support the conclusion that azide may be further modified in mammalian cells to an intermediate that is not genotoxic.     

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.     

Efficient production of androgenic doubled-haploid mutants in barley by the application of sodium azide to anther and microspore cultures

 The aim of this study was to establish a protocol for an efficient production of agronomical and/or physiological mutants from model (cvs. Igri and Cobra) and low-androgenic-responding (cv. Volga) cultivars of barley through the application of a mutagenic agent, sodium azide, to anthers and isolated microspores cultured in vitro. This technology offers the possibilities of screening for recessive mutants in the first generation, selecting for novel genotypes from very large haploid populations, avoiding chimerism and rapidly fixing selected genotypes as fertile true breeding lines. The mutagenic treatment, 10^–3–10^–5 M sodium azide, was applied during the anther induction pre-treatment or immediately after the microspore isolation procedure. Out of 616 M₂ doubled-haploid lines characterised under field conditions, a total of 63 morphological and developmental independent mutant lines were identified. The percentage of M₂ doubled-haploid lines carrying mutations per line analysed was 3.8% when 10^–4 M sodium azide was applied to anthers from the low-responding cv. Volga; this increased to 8.6% and 15.6% when 10^–5 and 10^–4 M sodium azide were applied to freshly isolated microspores from model cultivars. Received: 18 April 2000 / Revision received: 28 September 2000 / Accepted: 28 September 2000     

The very low mutagenic activity of sodium azide inArabidopsis thaliana

Sodium azide, reported to be a strong mutagen in barley, revealed a very weak mutagenic activity inArabidopsis.     

Function and organization of the G region of bacteriophage Mu; Host specificity determined by gene splicing

Chinese hamster ovary (CHO) cells were exposed to [³H]ethyl nitrosourea (ENU) or [³H]ethyl methanesulfonate (EMS) and the following DNA ethylation products were quantitated: 3- and 7-ethyladenine, O²-ethylcytosine, 3-, 7- and O⁶-ethylguanine, O²- and O⁴-ethyldeoxythymidine and the representative ethylated phosphodiester, deoxythymidylyl (3′–5′)ethyl-deoxythymidine. When mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus induced by these same treaments were compared with the observed ethylation products, mutations were found to correlate best with 3- and O⁶-ethylguanine. EMS induced approximately twice as many sister-chromatid exchanges (SCEs) as ENU at doses yielding equal mutation frequencies. When SCEs were indirectly compared with DNA ethylation products, 3-ethyladenine and ethylated phosphodiesters related best to SCE formation. Because mutation and SCE induction appear, at least in part, to be related to different DNA adducts, SCE induction by simple ethylating agents may not be a quantitative indicator of potentially mutagenic DNA damage.     

Mutagenic and chromosome-breaking effects of azide in barley and human leukocytes

Azide (10^?3 M, solution buffered at pH 3) is more effective in inducing mutations in embryonic shoots of seeds germinated between 8 and 16 h than in non-germinated seeds and in seeds germinated between 0 and 8 h and 16 to 28 h. This peak of chlorophyll-deficient seedling mutation frequency coincides with maximum frequencies of seedling lethals and DNA replication in the cells of the embryonic shoot. The mutation data suggest azide may only act on replicating DNA.Azide induced no chromosome-aberration frequencies significantly above controls in (1) embryonic shoots of barley seeds germinated for 8–12 h, (2) microsores of barley and (3) human leukocytes. It appears to be a point-mutation mutagen.     

Gene mutation and sister chromatid exchange in Chinese hamster cells

The mutation in hypoxanthine phosphoribosyl transferase gene and the induction of sister chromatid exchange (SCE) were comparatively studied treating Chinese hamster ovary cells with the mutagens ethylmethanesulphonate. N-methyl-N'-nitro-N-nitrosoguanidine, Mitomycin C and X-ray. All the agents exerted strong mutagenic effects and showed a dose-dependent relationship for the induction of SCEs.     

Dependency of the yield of sister-chromatid exchanges induced by alkylating agents on fixation time. Possible involvement of secondary lesions in sister-chromatid exchange induction

Chinese hamster V79 cells were pulse-treated (for 60 min) with various mutagens three, two or one cell cycles before fixation (treatment variants A, B and C, respectively) and the frequencies of induced SCEs were analysed and compared. The degree of increase in frequency of SCEs with dose in the treatment variants depended on the mutagen used. For the methylating agents MNU, MNNG and DMPNU, high yields of SCEs were obtained in the treatment variants A and B, and there was no difference in the efficiency with which these agents induced SCEs in these treatment variants. In the treatment variant C, however, no SCEs were induced with mutagen doses yielding a linear increase in SCE frequency in treatment variants A and B. A slight increase in SCE frequency in treatment variant C was observed only when relatively high doses of MNU or MNNG were applied. Like the above agents, EMS, ENU and MMS induced more SCEs in treatment variants A and B than in C, but for these agents treatment variant B was most effective and SCEs were induced over the entire dose range, also in treatment variant C. As opposed to the methylating and ethylating agents, MMC induced SCEs with high efficiency when treatment occurred one or two generations prior to fixation. There was no difference in SCE frequency between these treatment variants. MMC was completely ineffective for the induction of SCEs when treatment occurred three generations before fixation. The unexpectedly low SCE frequencies induced by the methylating and ethylating agents when treatment occurred one generation before fixation were not due to the exposure of cells to BrdU prior to mutagen treatment. From the results obtained, it is concluded that DNA methylation and ethylation lesions give rise to SCEs only with very low probability during the replication cycle after the lesion's induction, and that subsequent lesions produced during or after replication of the methylated or ethylated template (secondary lesions) are of prime importance for SCE formation after alkylation. For MMC, however, primary lesions seem to be most important for SCE induction.     

Evaluation of the mutagenic potential of the hair dye N-methyl-amino-2-nitro-4-N',N'-bis-(2-hydroxyethyl)-aminobenzene in a battery consisting of Drosophila and mammalian cytogenetic assays

The compound N-methyl-amino-2-nitro-4-N', N'-bis(2-hydroxyethyl)-aminobenzene was tested for mutagenic activity in the sex-linked recessive lethal test with Drosophila melanogaster, the induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) with Chinese hamster ovary (CHO) cells in vitro, and the micronucleus test with mouse bone-marrow cells in vivo. Consistently negative results were obtained with the 3 tests. The SCE tests gave positive results with prolonged treatments. It is concluded that reliable decisions about mutagenic activity cannot be based on the induction, in vitro, of SCEs alone.     

Effect of sodium selenite and methyl methanesulfonate or N-hydroxy-2-acetylaminofluorene co-exposure on sister-chromatid exchange production in human whole blood cultures

Sodium selenite (Na₂Se0₃) was tested for its sister-chromatid exchange (SCE)-inducing ability in human whole blood cultures and for the effect of its co-exposure with methyl methanesulfonate (MMS) or N-hydroxy-2-acetyl aminofluorene (N-OH-AAF) on SCE frequency. Long exposure times (77 h and 96 h) to 3.95 × 10^-6 M Na₂SeO₃ resulted in cell death as measured by mitotic indices, but mitotic figures were present after exposure to higher concentrations for a shorter time (19 h). High Na₂SeO₃ concentrations (7.90 × 10^?6 and 1.19 × 10^?5 M) resulted in a three-fold increase in the SCE frequency above background level (6–7 SCEs/cell). Exposure of lymphocytes to 1 × 10^?4 M MMS for the last 19 h of culture yielded an average SCE frequency of 30.17 ± 0.75 while a similar exposure to 2.7 × 10^?5 M N-OH-AAF resulted in 13.61 ± 0.43 SCEs/cell. Simultaneous addition of the high Na₂Se0₃ concentrations and MMS or N-OH-AAF to the cultures resulted in SCE frequencies that were 25–30% and 11–17%, respectively, below the sum of the SCE frequencies produced by the individual compounds.