Chromosome breakage induced by thymidylate stress in thymidylate synthase-negative mutants of mouse FM3A cells

In thymidylate synthase-negative mutants of mouse FM3A cells, thymidine starvation rapidly decreased mitotic activity and resulted in cell death (thymineless death). When the thymidine starvation was reversed by an addition of thymidine, mitotic activity was recovered, but the majority of mitotic cells exhibited extensive chromosome aberrations, including chromatid breaks, chromatid exchanges, and pulverizations. Autoradiographic examination revealed that chromosome instability was induced only in cells arrested in the S phase during thymidine starvation. Furthermore, the most sensitive sites to the chromosome-damaging effect appeared to be sites which had replicated just prior to thymidine starvation. During thymidine starvation, cells at other stages in the cell cycle were accumulated at the G1-S boundary, and they were insensitive to the chromosome-damaging effect. Thymidine starvation was also found to be recombinagenic. Complete removal from the medium of a thymidine analogue, 5-bromo-2'-deoxyuridine, resulted in a dramatic increase in the frequency of sister chromatid exchanges. These results support the view that thymidine starvation in mammalian cells results in thymineless death via induction of DNA double-strand breaks, leading to chromosome fragmentation as well as rearrangements in the cells synthesizing DNA.     

Mechanism of epipodophyllotoxin-induced cell death in poly(adenosine diphosphate-ribose) synthesis-deficient V79 Chinese hamster cell lines

Mutant Chinese hamster V79 cells selected for alterations in poly(ADP-ribose) metabolism were shown to be resistant to epipodophyllotoxin (VP-16)-induced cytotoxicity. Cell lines ADPRT 54 and ADPRT 351 have reduced activity of poly(ADP-ribose) polymerase. N2, N3, and N4 cell lines grow in the absence of nicotinamide, with total NAD levels 1.5-3% of those found in parental V79 cells grown in complete medium. When grown in complete medium, the mutant cell lines are 2.3- to 9.6-fold resistant to VP-16-induced cytotoxicity. All of the cell lines respond to VP-16 treatment by formation of protein-cross-linked DNA strand breaks. Upon drug removal, all the cell lines reverse the DNA strand breaks at similar rates. Our studies show a clear dissociation between induction of DNA strand breaks and cytotoxicity. However, there is a good correlation between drug-induced sister chromatid exchanges and cytotoxicity. Thus, N3 cells, with low levels of VP-16-induced sister chromatid exchanges, show reduced levels of cytotoxicity relative to parental V79 cells, despite the fact that both cell lines show similar levels of VP-16-induced protein-cross-linked DNA strand breaks. Additional studies show that the time course of VP-16-induced cytotoxicity correlated better with the time course of sister chromatid exchange formation than with protein-cross-linked DNA strand break formation. These studies provide strong support for the proposal that VP-16-induced cytotoxicity involves the induction of sister chromatid exchanges. Thus, we suggest that drug-induced stabilization of topoisomerase II-DNA complexes stimulates induction of sister chromatid exchanges, which consequently lead to cell death.     

Camptothecin hypersensitivity in poly(adenosine diphosphate-ribose) polymerase-deficient cell lines

Mutant cell lines, derived from the Chinese hamster V79 cell line, deficient in poly(adenosine diphosphate-ribose) polymerase activity, and previously shown to be resistant to topoisomerase II inhibitors, were found to be hypersensitive to camptothecin, a topoisomerase I inhibitor. In all the cell lines, camptothecin induced dose-dependent protein-associated DNA single-strand breaks and sister chromatid exchanges. The increased sensitivity to camptothecin-induced cytotoxicity was not associated with an increase in DNA single strand breaks or sister chromatid exchanges. These results suggest the absence of any direct causal relation between (1) camptothecin induced sister chromatid exchanges and cytotoxicity or (2) camptothecin induced DNA strand breaks and cytotoxicity. The hypersensitivity of these mutant cell lines to camptothecin suggests that poly(adenosine diphosphate-ribose) polymerase is involved with topoisomerase I in modulating camptothecin induced cytotoxicity.     

Cytogenetic studies on the in-vitro genotoxicity of 4-nitroquinoline-1-oxide on human-lymphocytes

Using the UV-mimetic mutagen 4-nitroquinoline-1-oxide (4NQO) to induce genetic damage in human cells (lymphoblastoid lines and primary cultures of peripheral blood samples), chromosome aberrations were induced by treating the cells with 4NQO at 1 X 10(-5) M for 24 h. The overwhelming majority of chromosome aberrations was of the chromatid (S + G2) type instead of the chromosome (G1) type. The most common chromatid aberrations were simple breaks, isochromatid breaks, and chromatid exchanges. When the number of chromatid breaks per cell value was used as a measurement for 4NQO sensitivity, lymphoblastoid cells from a xeroderma pigmentosum patient showed the highest sensitivity, followed by the cells of two melanoma patients and normal persons. These preliminary results suggest that 4NQO may be employed to develop an assay system as a biomarker for determining UV sensitivity in the human population.     

Sodium bisulfite induces morphological transformation of cultured Syrian hamster embryo cells but lacks the ability to induce detectable gene mutations, chromosome mutations or DNA damage

The ability of sodium bisulfite to induce morphological transformation and mutagenesis of cultured Syrian hamster embryo cells was examined. Treatment of the cells at neutral pH for 15 min with 5-20 mM sodium bisulfite resulted in a dose-dependent increase in cell transformation but no induction of gene mutations measured at two genetic loci. Treatment of the cells for 24 h increased the level of transformation, but also failed to induce chromosome aberrations, aneuploidy or DNA strand breaks in the cells. The only positive response for a DNA alteration was an increase in sister chromatid exchanges, but this effect was observed only with the longer exposures and not with the 15 min exposure, which also transformed the cells. Possible mechanisms for bisulfite-induced cell transformation are discussed.     

Cytogenetic effects of etoposide (VP-16) on human lymphocytes; with special reference to the relation between sister chromatid exchange and chromatid breakage

The effects of etoposide (VP-16) on sister chromatid exchange (SCE) and chromosome abnormalities were studied by using human peripheral lymphocytes. This drug produced a significant increase in the SCE frequency and chromosomal aberrations such as breaks, exchanges, and tetraploidy with or without endoreduplication. Analysis of the breakpoints of chromatid deletion with respect to their association with SCE in the metaphases from the second division demonstrated that VP-16-induced deletions arose with little dependence on the site of SCE. It is suggested that chromatid deletions induced by this drug derive from unrepaired chromatid breaks rather than from incomplete exchanges.     

271例正常人遗传安全性指标基准的综合研究

本文采用淋巴细胞染色体畸变、微粒、姐妹染色单体互换和细胞动力学参数对人群进行了遗传安全性综合研究。结果表明,年龄与染色体畸变、微核、姐妹染色单体互换、M_1、M_2均呈正相关。年龄与M_3、增殖率指数则呈负相关。男女性别间存在着显著差异。     

Sodium fluoride-induced morphological and neoplastic transformation, chromosome aberrations, sister chromatid exchanges, and unscheduled DNA synthesis in cultured syrian hamster embryo cells

The effects of exposure of early-passage Syrian hamster embryo cells in culture to sodium fluoride have been studied with respect to induction of morphological and neoplastic transformation, chromosome aberrations, sister chromatid exchanges, and unscheduled DNA synthesis. Exposure of Syrian hamster embryo cells to NaF concentrations between 75 and 125 micrograms/ml for 24 hr caused approximately 90 to 40% cell survival and resulted in a dose-dependent increase in the frequency of morphological transformation of the cells. Mass cultures of cells treated with NaF (75 or 100 micrograms/ml) for 24 hr, followed by continuous cultivation for 35 to 50 passages, developed the ability to grow in soft agar and to produce anaplastic fibrosarcomas when injected into newborn hamsters. In contrast, no morphological and neoplastic transformation was observed in untreated cells. Furthermore, a significant increase in chromosome aberrations at the chromatid level, sister chromatid exchanges, and unscheduled DNA synthesis was induced by NaF in a dose- and time-dependent manner. These results indicate that NaF is genotoxic and capable of inducing neoplastic transformation of Syrian hamster embryo cells in culture. A potential for carcinogenicity of this chemical, which is widely used by humans, is suggested. However, the carcinogenic risk of this chemical to humans may be reduced by factors regulating in vivo dose levels.     

Ataxia-telangiectasia-like Chinese hamster V79 cell mutants with radioresistant DNA synthesis, chromosomal instability, and normal DNA strand break repair

We have isolated three radiosensitive mutants (V-C4, V-E5, and V-G8) of the Chinese hamster V79 cell line which also show increased sensitivities to killing by bleomycin (approximately 2-5-fold) and ethyl methanesulfonate (approximately 2-fold). Genetic complementation analysis indicates that all three mutants belong to one complementation group. The mutants show a radioresistant DNA synthesis following X-ray irradiation when compared to wild-type V79 cells. Both the level and the rate of repair of DNA single- and double-strand breaks measured by DNA elution were similar to those observed in wild-type V79 cells. The level of spontaneously occurring chromosome aberrations in two of these mutants differs severalfold from the level observed in wild-type V-79 cells and in V-G8, to approximately 2- and 6-fold increase in V-E5 and V-C4, respectively. X-irradiation of the mutants resulted in consistently 3-4-fold higher levels of chromatid gaps, breaks, and exchanges than observed in wild-type V79 cells. In addition, G1 irradiation of the mutant cells yielded both chromosome and chromatid types of aberrations. The level and pattern of chromosomal aberrations induced by X-rays in V-C4, V-E5, and V-G8 are similar to those observed in ataxia-telangiectasia cells. These results indicate that our mutants represent the first rodent cell mutants which show phenotypic characteristics strongly resembling those in cells from ataxia-telangiectasia patients.     

Induction of chromosome breaks and sister chromatid exchanges in patients with Hodgkin's disease by two combination chemotherapy regimens of different leukemogenic potential.

The success of various combination chemotherapies in the treatment of cancer is compromised by their potential to cause secondary leukemia. Previous studies have suggested that the alkylating agents used in some regimens are the major etiological factor in these leukemias. In this study, we compared the abilities of two standard regimens used in the treatment of Hodgkin's disease to cause chromosome breaks and sister chromatid exchanges, the two most common types of chromosomal damage induced by alkylating agents. These regimens are MOPP [mechlorethamine-vincristine (Oncovin)-procarbazine-prednisone] and CVPP-ABDIC [cyclophosphamide-vinblastine-procarbazine-prednisone-doxorubicin (Adriamycin)-bleomycin-dacarbazine-1-(2-chloroethyl)-3-cyclohexyl-1- nitrosourea]. Our study demonstrated that (a) levels of spontaneous chromosome breaks and sister chromatid exchanges were low in untreated Hodgkin's disease patients; (b) significantly higher levels of these damages were induced in patients receiving eight cycles of CVPP-ABDIC, as compared with their pretreatment levels; (c) significantly elevated levels of sister chromatid exchanges, but not chromosome breaks, were induced in patients receiving two cycles of MOPP; and (d) no differences in the effect of these two regimens on cell cycle kinetics were observed. Although MOPP therapy has been reported to have higher rates of secondary leukemia than CVPP-ABDIC, our studies show that eight cycles of CVPP-ABDIC are more potent than two cycles of MOPP in inducing chromosome damage in patients during treatments.     

Repair of DNA damage induced by the novel nucleoside analogue CNDAG through homologous recombination

We postulate that the deoxyguanosine analogue CNDAG [9-(2-C-cyano-2-deoxy-1-β-d-arabino-pentofuranosyl)guanine] likely causes a single-strand break after incorporation into DNA, similar to the action of its cytosine congener CNDAC, and that subsequent DNA replication across the unrepaired nick would generate a double-strand break. This study aimed at identifying cellular responses and repair mechanisms for CNDAG prodrugs, 2-amino-9-(2-C-cyano-2-deoxy-1-β-d-arabino-pentofuranosyl)-6-methoxy purine (6-OMe) and 9-(2-C-cyano-2-deoxy-1-β-d-arabino-pentofuranosyl)-2,6-diaminopurine (6-NH2). Each compound is a substrate for adenosine deaminase, the action of which generates CNDAG. Growth inhibition assay, clonogenic survival assay, immunoblotting, and cytogenetic analyses (chromosomal aberrations and sister chromatid exchanges) were used to investigate the impact of CNDAG on cell lines. The 6-NH2 derivative was selectively potent in T cell malignant cell lines. Both prodrugs caused increased phosphorylation of ATM and its downstream substrates Chk1, Chk2, SMC1, NBS1, and H2AX, indicating activation of ATM-dependent DNA damage response pathways. In contrast, there was no increase in phosphorylation of DNA-PKcs, which participates in repair of double-strand breaks by non-homologous end-joining. Deficiency in ATM, RAD51D, XRCC3, BRCA2, and XPF, but not DNA-PK or p53, conferred significant clonogenic sensitivity to CNDAG or the prodrugs. Moreover, hamster cells lacking XPF acquired remarkably more chromosomal aberrations after incubation for two cell cycle times with CNDAG 6-NH2, compared to the wild type. Furthermore, CNDAG 6-NH2 induced greater levels of sister chromatid exchanges in wild-type cells exposed for two cycles than those for one cycle, consistent with increased double-strand breaks after a second S phase. CNDAG-induced double-strand breaks are repaired mainly through homologous recombination.     

Vulnerability of specific rat chromosomes to in vitro chemically induced damage.

The treatment of rat embryo secondary cultures with DMBA or DMBA-3H for 5, 9, or 24 h resulted in chromosome damage consisting mainly of chromatid type aberrations. There was an increase in the percentage of labelled nuclei and metaphases with increasing length of exposure. In terms of incidence of chromatid lesions, the largest telocentric chromosome (No. 2) was the most susceptible of the autosomes. Banding pattern analysis demonstrated that the region associated with negative band 2q24 of the No. 2 chromosome had the highest number of lesions. An increased accumulation of DMBA-3H label occurred in approximately the same chromatid area of a small fraction of cells exposed for either 5 or 9 h prior to mitosis. The complete loss of DMBA-3H chromosomal labelling after DNAse treatment suggests that the visible grains represent carcinogen-bound DNA. After DMBA and BrdUrd, there was an increase in the number of sister chromatid exchanges compared to controls treated with BrdUrd only; the location of the exchange points on chromosome No. 2 was similar in samples treated with either DMBA and BrdUrd or BrdUrd alone. Additional experiments with thymidine-3H showed that the non-random chromatid lesions on chromosome No. 2 may result from endogenous radiation from the incorporated tritium. These studies demonstrate that a specific chromosome may be affected by diverse agents and that chromatid lesions frequently occur at the site of sister chromatid exchanges.     

Increased incidence of chromosomal aberrations in peripheral lymphocytes of retired nickel workers

Chromosomal aberrations and sister chromatid exchanges wereanalysed in the peripheral lymphocytes of nine retired nickelrefinery workers 4 – 15 years after the retirement andcompared with 11 matched non-nickel exposed controls. None ofthe controls had previous occupations with known relation toinduction of chromosomal aberrations nor sister chromatid exchanges.The groups were equal as to socio-economic status and environmentalfactors other than the occupational ones, which could influencethe chromosome parameters, were to the largest possible extentexcluded. The nickel workers' previous occupational employmentinvolved exposure to inhalation of furnace dust of Ni₃S₂ andNiO or aerosols of NiCl₂ and NiSO₄. The concentration of nickelin the working atmospheres has been higher than 1.0 mg/m³ airand the exposure time more than 25 years. The retired nickelworkers showed an increased incidence of breaks (p <0.001)and gaps (p <0.05) but no difference in the incidence ofsister chromatid exchanges when compared with the controls.     

Differential sensitivity of Xeroderma pigmentosum cells of different repair capacities towards the chromosome breaking action of carcinogens and mutagens.

The sensitivity of cultured fibroblasts obtained from four unrelated Xeroderma pigmentosum patients (XP-K, XP-C, XP-E and XP-H), which showed different DNA repair levels, was examined. The frequency of metaphase plates with chromosome aberrations and the frequency of breaks and exchanges per chromosome complement were estimated following exposure to the carcinogens 4-nitroquinoline-1-oxide (4NQO),N-acetoxy-2-acetyl-aminofluorene (N-acetoxy-2-AAF), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), and to the mutagen daunomycin. The frequency of chromosome aberrations (breaks and exchanges) increased in the order (XP-K less than XP-C less than XP-E less than XP-H) with decreasing DNA repair capacity of the XP cells examined (XP-K greater than XP-C greater than XP-E greater than XP-H) following 4NQO and N-acetoxy-2-AAF. MNNG induced DNA repair synthesis and chromosome aberrations in the four XP cell types at levels comparable to those in fibroblasts of non-afflicted persons. Daunomycin triggered no DNA repair synthesis but induced similar frequencies of chromosome aberrations in the XP cells and controls. Heterozygous XP cells from parents of XP-K, XP-E and XP-C responded as control cells towards the three carcinogens and the mutagen used. Xeroderma pigmentosum can be considered to be an "induced" chromosome instability syndrome, in contrast to Bloom's syndrome or Fanconi's anaemia, which are "spontaneous" chromosome breakage syndromes according to German's definition.     

Chromosomal alterations in peripheral blood lymphocytes, urinary mutagenicity and excretion of polycyclic aromatic hydrocarbons in six psoriatic patients undergoing coal tar therapy

Six male non-smoking subjects treated for psoriasis with topical applications of pure coal tar or 4% coal tar-containing ointment were examined in order to assess the genotoxic risk associated with this type of therapy. Mutagenicity in urine samples collected before and during the coal tar therapy was evaluated in the plate incorporation assay on Salmonella typhimurium strain TA98 in the presence of S9 mix and beta-glucuronidase. Total urinary polycyclic aromatic hydrocarbon (PAH) levels were evaluated in parallel by high resolution gas chromatography/mass spectrometry. In addition, sister chromatid exchanges and chromosomal aberrations were also analysed in peripheral blood lymphocytes collected before, during and after the end of the coal tar applications. The results suggest that urinary mutagenicity levels as well as the frequencies of chromosome aberrations and sister chromatid exchanges in lymphocytes are related to the levels of exposure to coal tar. Moreover the kinetics of repair of chromosome damage in relation to different exposure levels and the capacity of the urinary mutagenicity assay to correctly identify the exposure to significant levels of PAH are discussed.     

The relationship between sister chromatid exchange, chromosome aberration and gene mutation induction by several reactive polycyclic hydrocarbon metabolites in cultured mammalian cells

The ability of three ultimate matabolites of benzo(a)-pyrene and of 7-bromomethylbenz(a)anthracene to induce 8-azaguanine mutants, sister-chromatid exchanges and chromosome aberrations has been investigated. 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene was shown to be an extremely efficient inducer of both mutants and sister-chromatid exchanges at 100% survival, whereas its geometrical isomer, 7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydro-benzo(a)pyrene and benzo(a)pyrene-4,5-oxide were comparatively weak. The potency of this compound, as both a mutagen and a sister-chromatid exchange inducer, gives further evidence that it may be the important carcinogenic metabolite of benzo(a)pyrene. 7-Bromomethylbenz(a)anthracene was a moderate mutagen and inducer of sister chromatid exchanges. Comparisons of the relative potencies of these four chemicals as inducers of both mutations and sister chromatid exchanges have indicated that these two phenomena are not directly related. The induction of sister chromatid exchanges also appears to be independent of the induction of chromosome aberrations.     

Assignment of the human gene for DNA polymerase alpha to the X chromosome

An interspecific cell hybrid was constructed between a temperature-sensitive mutant cell line of mouse FM3A cells (FT20-M6, a 6-thioguanine-resistant derivative of tsFT20) that has heat-labile DNA polymerase alpha and a human diploid fibroblast derived from a patient with the fragile X syndrome. After extensive segregation of the human chromosomes, a hybrid clone, named M6-39-11, was obtained that contained an X chromosome as the only human chromosome, was able to grow at the nonpermissive temperature and contained human DNA polymerase alpha. These data strongly suggest that the functional human gene for DNA polymerase alpha is located on the X chromosome.     

Molecular mechanisms of alkylation sensitivity in Indian muntjac cell lines

The responses of two Indian muntjac cell lines to two monofunctionalalkylating agents were investigated. An SV40-transformed line(SVM) had an increased sensitivity to cell killing when comparedto the other, euploid line (DM) after exposure both to methylnitrosourea (MNU) and to dimethylsulphate (DMS) and also exhibitedhigher frequencies of sister chromatid exchanges (SCEs) followingalkylation. The hypersensitivity of SVM to DMS correlates withthe defective repair of single-strand breaks that results inthe generation of long-lived breaks in the DNA following exposure,leading eventually to the formation of chromosome aberrations.In contrast no difference is seen in the formation of long-livedbreaks in the DNA of SVM and DM after treatment with biologicallyrelevant doses of MNU; in this case hypersensitivity may bedue to the loss of O⁶-alkylguanine-DNA-alkyltransferase activity.The conclusion that the hypersensitivities of SVM to MNU andto DMS have different molecular bases is supported by transfectionof SVM with plasmids containing the protein coding region ofthe Escherichia coli ada⁺ gene; subsequent expression withinthe cell corrects its hypersensitivity to the cytotoxic andSCE-inducing effects of MNU but has very little influence uponthe lethality, SCE induction or the repair of long-lived DNAstrand breaks after exposure to DMS.     

Production of sister chromatid exchanges by various cancer chemotherapeutic agents.

Various cancer chemotherapeutic agents have been examined for their ability to produce increases in sister chromatid exchanges. Those agents which had been shown previously to produce oncogenic transformation as well as chromosomal breaks also showed significant increases in sister chromatid exchanges. Those drugs which had not been shown to be oncogenic or clastogenic in cell culture produced no increases in sister chromatid exchanges. In general, concentrations which yielded increases in sister chromatid exchanges were considerably lower than those which had been shown previously to produce oncogenic transformation and chromosomal breakage. This was particularly true for the alkylating agents. Thus, we concur that examining increases in the production of sister chromatid exchanges may be an additional sensitive method for detecting potential mutagenic and/or oncogenic agents in our environment.