The frequency and distribution of isolabelling in Chinese hamster chromosomes after exposure to x-rays

2 experiments were carried out, following the same scheme. Chinese hamster cells (line CHEF-125) were cultivated for 4 h in the presence of tritiated thymidine ([³H]TdR) (Expt. 1, 0.5 μCi/ml; Expt. 2, 0.1 μCi/ml). After removal of the isotope, some of the cultures were X-irradiated in stage S-G₂ preceding M₁ (series S-G₂ I), others in stage S-G₂ preceding M₂ (series S-G₂ II).Analysis of the colchicine-C-anaphase of M₂ cells showed that in both experiments the X-rays produced an increase in the isolabelling regions only if given in the S-G₂ stage preceding M₁. On the other hand, the X-rays produced an increase in sister chromatid exchanges (SCEs) in both series of the second experiment. In the first experiment, where the controls had a high frequency of SCEs, no increase in SCEs was recorded in either of the two series treated with X-rays. The frequency of the isolabelling regions, in relation to the total labelling (Expt. 2), was 1.4% in the control series, 2.5% in series S-G₂ I and 1.3% in series S-G₂ II.Analysis of the distribution of the isolabelled regions along the chromosome showed that the regions furthest from the centromere were those most involved in this phenomenon and that, for the most part, the isolabelling was not directly associated with an exchange.The results have been interpreted, in accordance with the theory put forward in a previous work, to mean that SCEs and isolabelling regions are due to different phenomena, the former resulting from an exchange involving the whole chromatid, the latter from subchromatid exchanges probably involving a single polynucleotide strand.     

Comparison of sister chromatid exchanges from three successive cell cycles in Wallabia bicolor chromosomes

An autoradiographic analysis of tritiated thymidine labeled chromosomes of Wallabia bicolor at the second and third metaphases after label incorporations has shown that sister chromatid exchanges (SCE's) from the first and second cell cycles are less than as frequent as SCE's from the third cell cycle after label. Exchange levels per cell cycle estimated at the seconf division are under-estimated due to coincident exchanges. In both methaphases exchanges were largely distributed at random along Wallabia chromosomes with frequencies proportional to chromosome length. The ratio of twin: single SCE's in spontaneoulsy occuring tetraploid cells indicated the first cycle exchanges were marginally more frequent than second cycle exchanges.These data are compatible with exchange probabilities being equal and independent over divisions, but a component of exchanges reducing as tritium content in chromosomes decreases cannot be excluded. This findings that SCE's are primarily independent of tritium cannot be attributed to a saturation of sites for exchange and it is therefore probable that sister exchanges are, in part at least, spontaneous events in Wallabia chromosomes.     

Studies on induced aberrations in diplochromosomes of Chinese hamster cells

We have classified the commoner aberrations that can be scored in a diplochromosome. This chromosome configuration can be useful for a study of the mechanisms that underlie chromosome aberrations. Three experiments were done in which endoreduplicated Chinese hamster cell (strain CHEF-125) were irradiated at various stages in their cell cycle. The results showed good consistency in the three experiments and confirmed that the spatial arrangement of the chromosomes influences both the type and the frequency of the aberrations produced.     

Exogenous glutathione induces sister chromatid exchanges,clastogenicity and endoreduplication in V79-E Chinese hamster cells

Glutathione (GSH) dissolved in Eagle's MEM and added to cultures o of V79-E cells in concentrations between 2.5 × 10^–4 and 10^–3 moles/l for 1 h induces a dose-dependent cell cycle delay, sister chromatid exchanges and clastogenic damage. 7–8% of the metaphases showed endoreduplication at a recovery phase of 25 and 30 h after treatment with 10^–3 molesll GSH. Higher concentrations were lethal. The highest tolerated dose corresponds to the intracellular GSH level in V79-E cells. In the same range of concentrations, glutathione disulfide was inactive. Endoreduplication induction by GSH is G2-phase specific and endoreduplication metaphases show a reduced occurrence of single SCEs when extrapolated to the diploid complement. The adverse effects of GSH are independent of the presence of serum in the culture fluid but completely abolished when the treatment is performed in Hank's solution instead of MEM. The mechanism of genotoxicity of exogenous GSH is discussed but, at present, no pertinent explanation can be given.Abbreviations BUdR 5-bromodeoxyuridine - GSH glutathione - GSSG glutathione disulfide - SCE sister chromatid exchange     

Hyperthermic enhancement of chromosome damage and lack of effect on sister-chromatid exchanges induced by bleomycin in Chinese hamster cells in vitro.

Chinese hamster cells, M-3, were treated with BLM (1--4 micrograms/ml) for 30 min to 1 h at 37 degrees or 43 degrees C. After treatment, the cells were reincubated at 37 degrees until recovery. The material treated at 43 degrees showed increased damage expressed as chromosome and chromatid-type breaks and exchanges. Since the amount of BLM entering the cell at 37 degrees is supposedly similar to that which enters the cell at 43 degrees, the enhanced damage is the result of true synergism, and not the facilitation of the drug's entry into the cell.     

The effect of 0-methylhydroxylamine on the chromosomes of Chinese hamster cells]

0-methylhydroxylamine increases the frequency of all kinds of aberrations in Chinese hamster cells, except chromatid exchange. The percentage of aberrant metaphases and the average number of breakages per cell increases by 5,5-6 times as compared with the control. OMHA proves to be very effective inducer of chromosome aberrations. Since OMHA induces, except gene mutations, also chromosome aberrations, there are reasons to suppose that its effect is connected not only with transitions, but also with some other molecular mechanisms.     

The effect of caffeine on cytotoxicity, mutagenesis, and sister-chromatid exchanges in Chinese hamster cells treated with dihydrodiol epoxide derivatives of benzo[a]pyrene

The effect of caffeine on Chinese hamster V79 cells after treatment with the highly mutagenic (+/-)-7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-7,8,9,10-tetrahydrobenzo[a]pyrene, and the weaker mutagen (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, B[a]P-deiol-epoxide II, was studied at both the biological and molecular levels. Caffeine, at nontoxic dose levels, caused a synergistic reduction in cell survival induced by both isomers and also inhibited DNA elongation as measured by alkaline sucrose-gradient analysis of nascent DNA. However, caffeine did not affect the induction of either ouabain-resistant mutants or sister-chromatid exchanges by either isomer. These results suggest that enhanced cell killing by caffeine in benzo[a]pyrene-diol-epoxide treated V79 cells may be related to caffeine's inhibitory effect on DNA elongation. However, inhibition of DNA elongation by caffeine did not influence the resulting induced levels of mutagenesis or sister-chromatid exchanges.     

采用SCGE和SCE分析法研究姬松茸菌体多糖(Ab-Mp)对DNA损伤的抑制作用

热水浸提法提取姬松茸菌丝体多糖,每天以10mg/(kg bw)和20mg/(kg bw)两种剂量对小鼠进行灌胃,连续15d.腹腔注射环磷酰胺(CP),运用单细胞凝胶电泳(SCGE)法和姐妹染色体交换(SCE)分析法,研究Ab-Mp对染色体损伤的保护作用.结果表明,Ab-Mp降低了CP损伤后彗星细胞的比例,缩短了彗星尾长;降低了CP损伤的姐妹染色单体交换率.Ab-Mp对环磷酰胺诱发的DNA损伤具有拮抗作用.     

Synthesis and mutagenicity of 2-aryl-substitute ( o -hydroxy-, m -bromo-, o -methoxy-, o -nitro-phenyl or 4-pyridyl) benzothiazole derivatives on Salmonella typhimurium and human lymphocytes exposed in vitro

Derivatives of 2-aryl-substitute (o-hydroxy-, m-bromo-, o-methoxy-, o-nitro-phenyl or 4-pyridyl) benzothiazole were synthesized and tested for their mutagenicity in in vitro assays: (i) in the Ames test with Salmonella typhimurium TA98 and TA100 strains; and (ii) in the sister chromatid exchange (SCE) in cultured human lymphocytes. The four of compounds (BT-11, B-12, BT-14 and BT-15) caused statistically significant increase in revertant colonies of TA98 and TA100. Treatment of lymphocytes with compounds also caused a significant increase in SCE/cell in association with high levels and long exposure (300 μg/mL and 48 h) of the four compounds. It can be concluded that benzothiazole derivatives showed mutagenic activity and were also able to exert a genotoxic effect reducing both the replication index and mitotic index.     

The effects of high and low fluoride diets on the frequencies of sister chromatid exchanges

A recent report suggests that fluoride has mutagenic activity in mice. To examine the potential clastogenic effect of ingested fluoride, we examined the frequencies of baseline SCE and mitomycin C induced SCE as well as baseline chromsomal aberrations and cell-cycle kinetics in mice raised on high and low fluoride diets. The lack of significant differences in any of these parameters between the two groups of animals indicates that dietary fluoride is not clastogenic and supports the continued use of water fluoridation.     

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.     

The effects of high and low fluoride diets on the frequencies of sister chromatid exchanges

A recent report suggests that fluoride has mutagenic activity in mice. To examine the potential clastogenic effect of ingested fluoride, we examined the frequencies of baseline SCE and mitomycin C induced SCE as well as baseline chromosomal aberrations and cell-cycle kinetics in mice raised on high and low fluoride diets. The lack of significant differences in any of these parameters between the two groups of animals indicates that dietary fluoride is not clastogenic and supports the continued use of water fluoridation.     

The effect of caffeine on the duration of the mitotic phase cycle in CHO-K1 Chinese hamster cells, irradiated with x-rays

Alterations in the duration of mitotic cycle phases in X-irradiated Chinese hamster cells CHO K1 after caffeine (CF) treatment are studied. Delays in S- and G2-phases, induced by 1 and 5 Gr of X-irradiation, are partially or completely decreased by 1 mM or 5 mM CF, respectively. When CF is removed from the medium after irradiation, delays in S- and G2-phases are seen again, however long (0-12 hours) CF remains in the medium. The data obtained allow to suggest that since CF results in a radioresistant DNA synthesis, it may also postpone delays in S- and G2-phases, while cells are progressing through the cell cycle.     

Sequence of the novel joints present in the amplified DNA of N-phosphonacetyl-L-aspartate resistant Drosophila cells: Implication on the mechanisms of amplification in these cells

Summary— We have previously shown the presence, in the amplified DNA of a Drosophila cell line resistant to N-phosphonacetyl-L-aspartate (PALA), of two units of 150 kb and 120 kb respectively duplicated and amplified. The two joints (J1 and J2) linking these units as well as their respective wild-type counterparts have been sequenced. Sequence analysis indicates that a region of the Drosophila genome which corresponds to the proximal boundary of the 150 kb unit is common to both joints. In addition to this common region, the J1 junction possesses a 26-nucleotide sequence belonging to the J2 junction. This indicates that the J2 junction was the first formed, and that J1, therefore, results from recombination between J2 and a region of the wild-type genome 120 kb distal to J2. Sequence analysis also reveals that the joints result from illegitimate recombination between unrelated regions. AT-rich sequences, strand bias composition and putative topoisomerase I and II sites were found in at least one of the two parental sequences involved in the formation of the joints. On the basis of these results we can hypothesize that after two illegitimate recombinations between sister chromatids, leading first to J2 and then to J1, the amplification may have arisen by a series of homologous (unequal crossing-over) or illegitimate recombinations, or by an intrachromosomal rolling circle.     

Effects of styrene oxide on chromosome aberrations, sister chromatid exchange and hepatic drug biotransformation in chinese hamsters in vivo

In vivo inhalation exposure to styrene oxide (25, 50, 75 and 100 ppm) for 2, 4 or 20 days (25 ppm only) had no effects on chromosomal aberration rates or sister chromatid exchange (SCE) frequencies (BrdU/labelling performed in vitro) in the bone marrow cells of Chinese hamsters. The only positive response in aberration frequency was obtained when styrene oxide was injected in lethal concentration (500 mg/kg body weight, i.p.) into the animal. One animal out of six showed slightly elevated SCE values after this high dose. The response of the hepatic drug metabolizing enzymes to styrene oxide exposure was found to be rather weak, which may be due to rather high activity of epoxide hydratase in Chinese hamsters as compared to e.g. mouse.     

Sister chromatid exchange analysis in cultured primary lung,liver, and kidney cells of mice following in vivo exposure to vinyl carbamate

Summary Methods are described for the short-term culture (48 to 56 h) of lung, liver, and kidney cells from C57B1/6 mice. With these techniques, mice can be exposed in vivo to test compounds and the cells grown on cover glasses in the presence of 5-bromo-2′-deoxyuridine (BrdUrd) (5 μM) for analysis of sister chromatid exchange (SCE) and cell cycle kinetics. Mice exposed to vinyl carbamate (VC) ((10 to 60 mg/kg) by i.p. injection were used in the initial examination of this system. Cultured lung and kidney cells from epxosed animals (60 mg/kg) exhibited significant increases in SCE frequencies (approximately 3 to 5 times baseline); however, liver cells were much less responsive and showed less than a twofold increase over baseline SCE levels. Lung cultures initiated as long as 320 h after VC exposure (60 mg/kg) revealed a persistance of lesions leading to the formation of SCEs in vitro. This methodology permits analysis of cytogenetic damage in organs with very low mitotic activity after in vivo exposure to known or suspected genotoxicants. The research described in this article has been reviewed by the Health Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency nor does mention of trade names or commercial products constitute endorsement or recommendation for use.     

The effect of post-treatment with caffeine on survival and UV-induced mutation frequencies in Chinese hamster and mouse lymphoma cells in vitro

The effect of post-treatment with caffeine on the survival of a number of cell lines after UV-irradiation has been studied. The mouse lymphoma cell lines P388 and L5178YS were sensitized by caffeine but only after UV doses of 50 erg/mm² and above. V79 cells also showed sensitization by caffeine but CHO cells and two cell lines YS and YR derived from Yoshida sarcoma of rats, sensitive and resistant to UV radiation, respectively, showed no effect.P388 and V79 cells were both mutable by UV, and caffeine, when studied at a single expression time (42–48 h) and at a single dose level (0.5 M and 0.75 M, respectively) suppressed the UV-induced mutation frequency in both cell lines. L51788YS cells although sensitized by caffeine showed no increase in frequency of thymidine-resistant (TdR^r) colonies when irradiated with UV.On more detaled examination, caffeine was found to delay the expression of UV-induced mutations inV79 cells, and the delay was dependent on the dose of caffine used. The effect on expression time was less when caffeine was present 0–48 h than when it was present throughout the post-irradiation incubation period. Similar results were obtained in P388 cells.The data are discussed in relation to those of other workers and to the concept that caffeine inhibits an error prone post-replication repair process in mammalian cells     

Sister chromatid exchange studies for monitoring DNA damage and repair capacity after cytostatics in vitro and in lymphocytes of leukaemic patients under cytostatic therapy.

The effect of various cytostatic drugs was studied on the frequency of sister chromatid exchanges (SCEs) in vitro and in PHA-stimulated lymphocytes of leukaemic patients under cytostatic therapy. The lymphocyte system is a sensitive one for the detection of DNA damage after administration of cytostatic drugs in vitro. Mitomycin C, busulphan, vincristine, chlorambucil, cytosine arabinoside, cyclophosphamide and lycurim were tested. All except cyclophosphamide induced high frequencies of SCEs in the first mitosis after their administration. The experiments with PHA-stimulated lymphocytes in vivo from patients treated with cytostatics showed that cytosine arabinoside, in combination with thioguanine, did not induce higher frequencies of SCEs, whereas in patients who were treated with cyclophosphamide alone or in combination with other cytostatic drugs, there was a higher incidence of SCEs during treatment. About 10 days after the termination of the treatment the elevated freuqencies of SCEs returned to the initial level. After administration of some mutagens, especially alkylating agents in vivo, the lymphocyte system can be used to assess induced DNA repair by continuously monitoring for SCEs.     

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.