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1.
A H Uggla 《Mutation research》1990,231(2):233-242
Chinese hamster ovary (CHO) cells were synchronized by mitotic shake-off, treated with the fluorochrome acridine orange (AO; 0.5 micrograms/ml), washed free of excess dye and subsequently exposed to visible light (2 X 40 W/8 Wm-2). The light exposure was performed on cells in the G1, G1/S, S or G2 phase of the cell cycle. AO + light induced high frequencies of aberration in the S phase and even higher in the G1 phase. The aberrations observed were all of the chromatid type. The chromosome-type aberrations (dicentrics, rings) obtained when cells in the G1 phase were exposed to X-rays were not found after corresponding treatments with AO + light. With the exception of an increased frequency of gaps, no chromosomal aberrations were induced in G2-phase cells. Sister-chromatid exchanges were efficiently produced by the photodynamic system in the G1, G1/S and S phase of the cell cycle. In other experiments, AO-treated unsynchronized CHO cells were exposed to light in the presence of the hydroxyl radical scavengers mannitol (100 mM) and 5-dimethyl thiourea (100 mM). In parallel experiments these scavengers were found to reduce markedly the chromosome breaking effects by X-rays but had no influence on the photodynamic induction of chromosomal alterations. The results presented show that the visible light-induced chromosomal alterations in CHO cells sensitized with the fluorochrome AO are obtained by an S-dependent mechanism. Furthermore, the results indicate that the hydroxyl free radical does not play a major role in the production of chromosomal alterations by AO + light.  相似文献   

2.
An assay for visualization of DNA loops undergoing supercoiling changes has been developed. The assay utilizes the fluorescent dye, propidium iodide (PI), which intercalates into the DNA and under the proper conditions causes the supercoiling status of the DNA to change. Thus, the DNA can be seen as a fluorescent halo that changes diameter with PI concentration. At low PI concentrations (0-7.5 micrograms/ml) the supercoils are relaxed with increasing PI, while at higher PI concentrations (7.50-50 micrograms/ml) supercoils in the opposite winding sense are rewound with increasing PI. When HeLa cells were irradiated with 1-20 Gy of 137Cs gamma-rays, the ability to rewind the DNA supercoils was inhibited in a dose-dependent manner, presumably because of the presence of radiation-induced DNA strand breakage, which removed the topological constraints on the DNA loops. These lesions were repaired rapidly during post-irradiation incubation. The ability of the DNA loops to be rewound was restored within 8 min after 10 Gy of gamma-irradiation, such that no difference from control cells could be detected. The half-time for repair of the radiation-induced lesions that inhibit DNA rewinding was similar to that for repair of DNA single strand breaks. The assay has certain advantages over current methods for assaying DNA damage in that it involves measurement of single cells and it does not require the DNA to be labeled with radioactive precursors.  相似文献   

3.
Neutral sucrose gradients of supercoiled DNA (phiX-174 RF I) were used to measure the in vitro production of strand breaks by the carcinogen, N-acetoxy-2-acetylaminofluorene (AcO-AAF). Treatment with AcO-AAF in 10% dimethyl sulfoxide did not directly yield strand breaks. Breaks in relatively low yield appeared after alkali treatment (pH 13, 60 min) of the RF I previously reacted with AcO-AAF. The DNA treated with AcO-AAF was sensitive to single-strand breakage by 303 nm near-ultraviolet light under neutral conditions. The greater the prior AcO-AAF treatment, the greater the sensitivity to 303 nm light. Post-irradiation alkali treatment greatly enhanced the light-induced strand breakage.  相似文献   

4.
A possibility of formation of breaks in DNA chains under the influence of visible laser radiation (lambda = 532 nm) on DNA--dye-intercalator complexes has been demonstrated theoretically and corraborated experimentally. The most probable mechanisms of the induction of breaks in DNA chains are discussed. Theoretical estimates and the experiments have been performed on complexes of DNA of lambda-phage with acridine orange dye (AO) under irradiation of these complexes by picosecond (tau p = 30 ps) pulses of second harmonic YAG:Nd laser. A strong effect of the formation of double-strand breaks is observed at light intensity of some GW/sm2 and dose of several hundred pulses.  相似文献   

5.
Radiation induced damage, i.e., the induction of DNA strand breaks, was studied on the level of single, unlabeled cells. DNA strand breaks were determined by direct partial alkaline unwinding in intact cell nuclei followed by staining with acridine orange, a development of a proposal first described by B. Rydberg (Int J Radiat Biol 46:521-527, 1984). The ratio of green fluorescence (double-stranded DNA) to red fluorescence (single-stranded DNA) in single cells was taken as a measure of DNA strand breaks. CHO-K1 and M3-1 cells irradiated with X-rays show a dose dependent induction of DNA strand breaks. Incubation at 37 degrees C after irradiation leads to repair of breaks. A repair halflife of about 10-11 min can be determined. Cell cycle specific differences in the induction of DNA strand breaks or repair behavior are not detectable at the resolution achieved so far. This new method offers two major advantages: the resolution of DNA damage and repair on the level of single cells and no need for labeling, thereby allowing for DNA damage and repair to be assessed in biopsy material from tumor patients.  相似文献   

6.
Illumination with red light of murine L929 fibroblasts that had been sensitized with haematoporphyrin derivative caused DNA single-strand breaks after a lag time of about 20 min, as revealed by alkaline elution. The cells appeared not to be capable of recovering from this damage. The photodynamic effect of haematoporphyrin derivative on DNA repair was assessed by monitoring the repair kinetics of DNA damage induced by either X-rays, u.v. light (254 nm) or methyl methanesulphonate treatment subsequent to a non-DNA-damaging photodynamic treatment with haematoporphyrin derivative. On 'post-incubation', the normally rapid repair of X-ray-induced DNA strand breaks did not occur, whereas with u.v. light and methyl methanesulphonate treatment after photodynamic treatment prolonged post-incubation resulted in an increase in the number of strand breaks rather than the normally observed decrease. This clearly shows that, after a photodynamic treatment with haematoporphyrin derivative that itself did not cause strand breaks, excision repair in L929 cells is severely inhibited at a stage beyond the incision step.  相似文献   

7.
X-ray-induced DNA base damage can be detected using endonuclease III and formamidopyrimidine-glycosylase, which create DNA strand breaks at enzyme-sensitive sites. Strand breaks can then be measured with excellent sensitivity using the alkaline comet assay, a single-cell gel electrophoresis method that detects DNA damage in individual cells. In using this approach to measure the oxygen enhancement ratio (OER) for radiation-induced base damage, we observed that the number of enzyme-sensitive sites increased with dose up to 4 Gy in air and 12 Gy in hypoxic WIL2NS cells. After rejoining of radiation-induced strand breaks, base damage was detected more easily after higher doses. The number of radiation-induced enzyme-sensitive sites was similar under both air and nitrogen. Base damage produced by hydrogen peroxide and 4-nitroquinoline-N-oxide (4NQO) was also measured. Results with hydrogen peroxide (20 min at 4 degrees C) were similar to those observed for X rays, indicating that enzyme-sensitive sites could be detected most efficiently when few direct strand breaks were present. Removing DNA-associated proteins before irradiation did not affect the ability to detect base damage. Base damage produced by 4NQO (30 min at 37 degrees C) was readily apparent after treatment with low concentrations of the drug when few 4NQO-induced strand breaks were present, but the detection sensitivity decreased rapidly as direct strand breaks increased after treatment with higher concentrations. We conclude that: (1) the OER for base damage is approximately 1.0, and (2) the presence of direct DNA strand breaks (>2000-4000 per cell) prevents accurate detection of base damage measured as enzyme-sensitive sites with the alkaline comet method.  相似文献   

8.
1-Nitropyrene and its chemically synthesised derivatives were investigated for their cytotoxicity and ability to induce DNA-strand breaks in Chinese hamster lung fibroblasts. Both 1-nitrosopyrene (0.25-60 micrograms/ml) and 1-aminopyrene (0.25-25 micrograms/ml) were cytotoxic, and induced the formation of DNA lesions, which were measured as DNA single-strand breaks after sedimentation in alkaline sucrose-density gradients. Higher doses of 1-aminopyrene (25-60 micrograms/ml) inhibited the formation of DNA single-strand breaks. 1-Nitropyrene was not toxic (0.25-60 micrograms/ml) and induced low levels of detectable DNA strand breaks, whilst N-acetyl-1-aminopyrene was inactive. The post-mitochondrial supernatant fraction of Aroclor-induced rat-liver containing 4 mM NADPH (S9 mix) did not promote the activation of 1-nitropyrene. In fact DNA strand breaks induced by either 1-nitropyrene or 1-nitrosopyrene was abolished in the presence of S9 mix. The 1-nitropyrene reduced intermediate, N-hydroxy-1-aminopyrene was synthesised by the reduction of 1-nitrosopyrene with ascorbic acid. In the presence of ascorbic acid, 1-nitrosopyrene caused a 5-fold increase in the number of DNA single-strand breaks when compared to cells treated with 1-nitrosopyrene alone. The results are discussed in terms of the metabolic activation of 1-nitropyrene and 1-aminopyrene in Chinese hamster lung cells.  相似文献   

9.
DNA strand breaks can be detected with great sensitivity by exposing calf thymus DNA to alkaline solutions and monitoring the rate of strand unwinding. Fluorometric analysis of DNA unwinding (FADU) is a reliable method for detecting single-strand DNA breaks as an index of DNA damage induced by photosensitizer.m-Chloroperbenzoic acid (CPBA) was used as a photosensitizer in the photodamage of calf thymus DNA. When DNA is exposed to ionizing radiation, the radicals produced in the irradiated sample modify the base-pair regions of the double strands. The protective action of copper salt, Schiff base [ethylene diamine with ethyl acetate](L) and its Cu(II) complex (Cu(7) L Cl(14)) against DNA damage photoinduced by CPBA was studied using ethidium bromide as a fluorescent probe. Treatment of DNA with 5, 10, 50, 100, or 200 microM CPBA produced 75%, 48%, 38%, 32% and 30% double-stranded DNA remaining, respectively after 30 min of alkaline treatment at 15 degrees C. Treatment of calf thymus DNA irradiated with CPBA with a dose of 1 mM [Cu(7) L Cl(14)] produced 96% double-stranded remaining protection under the same conditions compared with irradiated DNA without addition of Cu(II) complex of Schiff base.  相似文献   

10.
E Boye  W Khnlein    K Skarstad 《Nucleic acids research》1984,12(21):8281-8291
DNA strand breaks induced by Neocarzinostatin in Escherichia coli cells have been characterized. Radioactively labeled phage lambda DNA was introduced into lysogenic host bacteria allowing the phage DNA to circularize into superhelical molecules. After drug treatment DNA single- and double-strand breaks were measured independently after neutral sucrose gradient sedimentation. The presence of alkali-labile lesions was measured in parallel in alkaline sucrose gradients. The cell envelope provided an efficient protection towards the drug, since no strand breaks were detected unless the cells were made permeable with toluene or with hypotonic Tris buffer. In permeable cells, no double strand breaks could be detected, even at high NCS concentration (100 micrograms/ml). Induction of single-strand breaks leveled off after 15 min at 20 degrees C in the presence of 2 mM mercaptoethanol. Exposure to 0.3N NaOH doubled the number of strand breaks. No enzymatic repair of the breaks could be observed.  相似文献   

11.
Synthetic eumelanin prepared by autooxidation of D,L-DOPA causes DNA strand breaks, as determined by alkaline elution after cell lysis with detergent and proteolysis, in B16CL4 mouse melanoma cells. The melanin is toxic to the cells in the range of doses that causes strand breaks. When the melanin was incubated with the cells at 37 degrees C in tissue culture medium, it was maximally effective after 15 to 20 min at causing strand breaks in the DNA. The extent of damage is concentration dependent, but the effect plateaus at 1 mg/ml. The nature of the interaction of the cellular DNA with melanin is consistent with strand breaks, not DNA-DNA crosslinks. The strand break damage is repaired, even in the continued presence of melanin, but repair is more rapid if the cells are washed and the melanin is removed. The form of the melanin is important for obtaining the effect. Sonication for 3 min abrogates the effect to a considerable extent, and repeated cycles of sonication can completely destroy the activity. Lost activity returns slowly with storage at 4 degrees C. Melanin is more effective at damaging DNA in a protein-free medium. It is also DNA-damaging at 4 degrees C, but less so than at 37 degrees C. Preliminary studies indicate that the strand breaks caused by melanin are additive with those caused by ionizing radiation. The extent of DNA strand breaks and alkali-labile sites caused by several other melanins was also determined. Some melanins did not cause frank strand breaks, but were active in causing alkali-labile sites.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

12.
The tumor suppressor protein p53 serves as a critical regulator of a G1 cell cycle checkpoint and of apoptosis following exposure of cells to DNA-damaging agents. The mechanism by which DNA-damaging agents elevate p53 protein levels to trigger G1/S arrest or cell death remains to be elucidated. In fact, whether damage to the DNA template itself participates in transducing the signal leading to p53 induction has not yet been demonstrated. We exposed human cell lines containing wild-type p53 alleles to several different DNA-damaging agents and found that agents which rapidly induce DNA strand breaks, such as ionizing radiation, bleomycin, and DNA topoisomerase-targeted drugs, rapidly triggered p53 protein elevations. In addition, we determined that camptothecin-stimulated trapping of topoisomerase I-DNA complexes was not sufficient to elevate p53 protein levels; rather, replication-associated DNA strand breaks were required. Furthermore, treatment of cells with the antimetabolite N(phosphonoacetyl)-L-aspartate (PALA) did not cause rapid p53 protein increases but resulted in delayed increases in p53 protein levels temporally correlated with the appearance of DNA strand breaks. Finally, we concluded that DNA strand breaks were sufficient for initiating p53-dependent signal transduction after finding that introduction of nucleases into cells by electroporation stimulated rapid p53 protein elevations. While DNA strand breaks appeared to be capable of triggering p53 induction, DNA lesions other than strand breaks did not. Exposure of normal cells and excision repair-deficient xeroderma pigmentosum cells to low doses of UV light, under conditions in which thymine dimers appear but DNA replication-associated strand breaks were prevented, resulted in p53 induction attributable to DNA strand breaks associated with excision repair. Our data indicate that DNA strand breaks are sufficient and probably necessary for p53 induction in cells with wild-type p53 alleles exposed to DNA-damaging agents.  相似文献   

13.
The role of UV-induced DNA lesions and their repair in the formation of chromosomal aberrations in the xrs mutant cell lines xrs 5 and xrs 6 and their wild-type counterpart, CHO-K1 cells, were studied. The extent of induction of DNA single-strand breaks (SSBs) and DNA double-strand breaks (DSBs) due to UV irradiation in the presence or absence of 1-beta-D-arabinofuranosylcytosine (ara-C) and hydroxyurea (HU) was determined using the alkaline and neutral elution methods. Results of these experiments were compared with the frequencies of induced chromosomal aberrations in UV-irradiated G1 cells treated under similar conditions. Xrs 6 cells showed a defect in their ability to perform the incision step of nucleotide repair after UV irradiation. Accumulation of breaks 2 h after UV irradiation in xrs 6 cells in the presence of HU and ara-C remained at the level of incision breaks estimated after 20 min, which was about 35% of that found in wild-type CHO-K1 cells. In UV-irradiated CHO-K1 and xrs 5 cells, more incision breaks were present after 2 h compared with 20 min post-treatment with ara-C, a further increase was evident when HU was added to the combined treatment. The level of incision breaks induced under these conditions in xrs 5 was about 80% of that observed in CHO-K1 cells. UV irradiation itself did not induce any detectable DNA strand breaks. Accumulation of SSBs in UV-irradiated cells post-treated with ara-C and HU coincides with the increase in the frequency of chromosomal aberrations. These data suggest that accumulated SSBs when converted to DSBs in G1 give rise to chromosome-type aberrations, whereas strand breaks persisting until S-phase result in chromatid-type aberrations. Xrs 6 appeared to be the first ionizing-radiation-sensitive mutant with a partial defect in the incision step of DNA repair of UV-induced damage.  相似文献   

14.
Particulate lead chromate is a highly water-insoluble cytotoxic and carcinogenic agent, but its mechanism of action remains obscure. We investigated its effects on DNA damage in CHO cells after a 24-h exposure using alkaline or neutral filter elution and cytogenetic studies. Concentrations (0.08, 0.4 and 0.8 micrograms/cm2), which reduced the colony-forming efficiency of CHO cells to 94, 50 and 10%, respectively, produced dose-dependent DNA single-strand breaks and DNA-protein crosslinks, but no DNA double-strand breaks or DNA-DNA crosslinks were observed. The single-strand breaks were absent from cells given a 24-h recovery period after removal of the treatment medium, even though most of the particles remained adhered to cells and to the culture dish. In contrast, both the DNA-protein crosslinks and chromosomal aberrations persisted even after the 24-h recovery period. These results suggest that the mechanism of the particle-induced early DNA single-strand breaks may be different from DNA-protein crosslinks and the lesions leading to chromosomal aberrations, or alternatively, that the repair of single-strand breaks is more efficient than the repair of DNA-protein crosslinks in the unavoidable continuing presence of carcinogen. These results also suggest that the chromosome damage may be related to the persistent DNA-protein crosslinks, and further confirm the genotoxic activity of carcinogenic lead chromate particles.  相似文献   

15.
Elaboration of cellular DNA breaks by hydroperoxides.   总被引:2,自引:0,他引:2  
Cellular damage produced by ionizing radiation and peroxides, hydrogen peroxide (HOOH) and the organic peroxides tert-butyl (tBuOOH) or cumene hydroperoxide (CuOOH) were compared. DNA breaks, toxicity, malondialdehyde production, and the rate of peroxide disappearance were measured in a human adenocarcinoma cell line (A549). The alkaline and neutral filter elution assays were used to quantitate the kinetics of single and double strand break formation and repair (SSB and DSB), respectively. Peroxides, at 0.01-1.0 mM, produce multiphasic dose response curves for both toxicity and DNA SSBs. Radiation, 1-6 Gy, produced a shouldered survival curve, and both DNA SSB and DSBs produced in cells x-rayed on ice were nearly linear with dose. The peroxides produced more SSBs than radiation at equitoxic doses. X-ray induced DNA single strand breaks were rejoined rapidly by cells at 37 degrees C with approximately 80% of initial damage repaired in 20 min. Peroxide induced SSBs were maximal after 15 min at 37 degrees C. Rejoining proceeded thereafter, but at a rate less than for x-ray induced strand breaks. Significant DNA DSBs could not be achieved by peroxides even at concentrations 50-fold higher than required to produce SSBs. HOOH treatment of DNA on filters following cell lysis and proteolysis produced SSBs. CuOOH and tBuOOH produced no SSBs in lysed cell DNA. None of the peroxides produced DSBs when incubated with lysed cell DNA. Malondialdehyde was released from cells incubated with organic hydroperoxides, but not HOOH, nor up to 40 Gy of x-rays. HOOH was metabolized three times faster than the organic peroxides. The overall results demonstrate the necessity for a metabolically active cell environment to elaborate maximal DNA strand breaks and cell death at hydroperoxide concentrations of 10(-4) or greater, but prevent strand breaks and stimulate cell growth at 10(-5) M.  相似文献   

16.
Quantitation of single- and double-strand DNA breaks in vitro and in vivo   总被引:4,自引:0,他引:4  
This communication describes a rapid and convenient procedure for quantitation of strand breaks in bacterial DNA, both in vitro and in vivo, using agarose gel electrophoresis. The electrophoretic determination of single strand breaks is carried out in alkaline medium, followed by renaturation of the gel and intercalation of the fluorescent dye, ethidium bromide. Double-strand breaks are determined by electrophoresis in neutral medium containing the dye. The distribution of DNA fragment sizes, the determination of the number-average molecular weight, the quantitation of the average number of DNA breaks per molecule, and the ratio between the single- and double-strand breaks are evaluated from microdensitometric scanning of the gels. The application of this analysis to damage caused by a combination of ascorbate and copper is demonstrated.  相似文献   

17.
R D Snyder 《Mutation research》1988,193(3):237-246
The ability of 6 metal salts to induce DNA damage in human diploid fibroblasts was examined. Cadmium, magnesium, manganese, chromium(VI), zinc and selenite were all shown to induce DNA strand breaks as measured by two independent assays. DNA strand breaks were repaired within 2-4 h after removal of metal and this repair appeared not to be sensitive to "long-patch" repair inhibitors. With the exception of selenite, metal-induced DNA damage appeared to be mediated via the formation of active oxygen species since oxygen scavengers when administered simultaneously with the metal, antagonized strand break formation. Selenite-induced DNA damage (as previously reported) was dependent on the formation of a selenite-glutathione conjugant and was not affected by oxygen radical scavengers. Scavenger treatment did not enhance cloning ability of metal-treated cells suggesting that DNA strand breaks may not be important in metal-induced cytotoxicity.  相似文献   

18.
Strand breakages of mammalian cellular chromosomal DNA with aromatic reductones were ascertained by use of a cultured cell strain of the rat fetal lung (RFL). The mode of the breakages was investigated by ultracentrifugal analyses. The reductones induced the breakages of the cellular DNA in two different fashions; one is single strand breaks and another double strand breaks. Although the single strand breaks were rapidly repaired, double strand breaks were only partially repaired. Both breaks were not cytocidal. Some physiological alterations were observed to follow the strand breaks.  相似文献   

19.
Single-stranded DNA has been speculated to be the initial signal in the DNA damage signaling pathway. We showed that introduction of single-stranded DNA with diverse sequences into mammalian cells induced DNA damage as well as apoptosis signals. Like DNA damaging agents, single-stranded DNA up-regulated p53 and activated the nuclear kinase ataxia telangiectasia mutant (ATM) as evidenced by phosphorylation of histone 2AX, an endogenous ATM substrate. Single-stranded DNA also triggered apoptosis as evidenced by the formation of caspase-dependent chromosomal DNA strand breaks, cytochrome c release, and increase in reactive oxygen species production. Moreover, single-stranded DNA-induced apoptosis was reduced significantly in p53 null cells and in cells treated with ATM small interfering RNA. These results suggest that single-stranded DNA may act upstream of ATM/p53 in DNA damage signaling.  相似文献   

20.
端粒是位于真核细胞染色体末端的DNA-蛋白质复合体,在维持染色体稳定上起着重要的作用,并且与细胞的衰老、癌变有着密切的关系。本实验观察了DNA依赖性蛋白激酶(DNA-dependent protein kinase,DNA-PK)抑制剂-渥曼青霉素(wortmannin,WM)对H2O2诱导的HeLa细胞端粒DNA链断裂重连接效应。结果表明WM能够显著地抑制H2O2诱导的HeLa细胞端粒DNA链断裂后的重连接作用,提示DNA-PK参与了端粒DNA链断裂损伤的修复过程。  相似文献   

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