BCNU-induced sister chromatid exchanges are increased by X irradiation

We have studied the effect on sister chromatid exchange (SCE) induction in 9L rat brain tumor cells caused by combination treatment with BCNU and X rays. Over the dose and concentration ranges used in these experiments, BCNU induced relatively large numbers of SCEs, while X rays induced few SCEs. When cells were X irradiated immediately after BCNU treatment, the number of SCEs induced was greater than the number of SCEs expected by adding the number of SCEs induced by each agent alone; the number of SCEs induced as a result of this BCNU-X-ray interaction increased as the concentration of BCNU and/or dose of X rays increased. When the addition of bromodeoxyuridine was delayed from 0 to 16 hr after BCNU treatment, the number of SCEs induced declined to control levels by 16 hr. If X irradiation was delayed for up to 16 hr after BCNU treatment the same pattern of decrease was observed; the number of SCEs induced at each time point, however, was greater than that induced by BCNU and X rays alone. X irradiation from 0-16 hr before BCNU treatment produced the same number of SCEs as that produced by BCNU alone. Thus the SCE assay is capable of detecting a drug-X-ray interaction in mammalian cells and provides a sensitive means of studying the sequencing and timing that leads to the interaction.     

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 (Na2SeO3) 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-acetylaminofluorene (N-OH-AAF) on SCE frequency. Long exposure times (77 h and 96 h) to 3.95 X 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 X 10(-6) and 1.19 X 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 X 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 X 10(-5) M N-OH-AAF resulted in 13.61 +/- 0.43 SCEs/cell. Simultaneous addition of the high Na2SeO3 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.     

Cytogenetic analysis of human peripheral blood lymphocytes in culture exposed in vitro to styrene and styrene oxide

Styrene and styrene oxide mutagenicity was tested in cultured human lymphocytes treated in vitro with various concentrations of test agents. Styrene alone was found mutagenic at the highest concentration used (5 X 10(-4) mol. l-1, combined with the alkylating agent THIO-TEPA it did not affect the chromosome aberration yield. Exposure to styrene oxide gave a positive result showing a clear-cut dose-effect relationship within the concentration range 5 X 10(-6) to 1 X 10(-3) mol. l-1. In combination with THIO-TEPA its effect on chromosome aberration yields was additive. Styrene oxide proved also to be a very potent inducer of sister chromatid exchanges (SCE) within the concentration range 5 X 10(-6) to 1 X 10(-3) mol. l-1 tested. Combined with THIO-TEPA it exhibited a distinct additive effect in the production of SCEs.     

In vitro SCE discrepancy between embryonic and extraembryonic mouse tissues

In vitro sister-chromatid exchange (SCE) background levels and cytokinetics were compared in embryonic (whole embryo cell suspensions) and extraembryonic (yolk sac and amnion, placenta) cells of inbred and outbred strains at various gestational stages (days 12-17). Results indicate a tissue origin (embryonal, extraembryonal) related variation in the formation of baseline SCE frequencies and cytokinetics. The significant higher SCE levels in extraembryonic tissues (maximum increase of 2 X the background values of the embryo cells) were independent of mouse strain and gestational stage. An average of 4-5 SCEs/cell in embryo cells is contrasted by 7-9 SCEs/cell in extraembryo cells. Mitotic index was generally lower and average generation time longer (by 2-3 h) in extraembryonic tissue cells. No significant differences in SCE frequencies and no changes in cytokinetics were detected at the BrdU concentrations used (1.2-4.8 micrograms/ml). The reason for the inter-tissue differences in baseline SCE is still not clear.     

A unique human mutant B-lymphoblastoid cell line (ataxia telangiectasia) which exhibits increased siste-chromatid exchange retaining hypersensitivity to neocarzinostatin and bleomycin

Chromosome aberrations and sister-chromatid exchanges (SCEs) were examined in 4 ataxia telangiectasia (AT)-derived B-lymphoblastoid cell lines (B-LCLs) (AT-S, AT-SHI, AT-SHI B13A and AsHa) following treatments with neocarzinostatin (NCS) and bleomycin. All of these cell lines exhibited extremely high frequencies of chromosome aberrations with the NCS and bleomycin treatments. Among them, AsHa, a mutant B-LCL originating from an AT patient, showed high frequencies of SCEs under high bromodeoxyuridine (BrdU) concentrations retaining hypersensitivity to NCS and bleomycin with regard to chromosome aberrations. A clear BrdU dose-dependent increase in SCEs (9.85 SCEs/cell at 40 μg/ml, 36.65 SCEs/cell at 100 μg/ml on average) in this mutant was observed. When AsHa mutant cells were treated with NCS (0.02 μg/ml) and/or bleomycin (5.0 μg/ml) under 40 μg/ml BrdU (minimum BrdU concentration for sister-chromatid differential staining), SCE levels increased from 9.85 (baseline level) to 21.1 with NCS and 20.5 with bleomycin, in a dose-dependent manner. These observations indicate that AsHa is a unique AT-derived mutant cell clone with a high SCE character retaining the original hypersensitivity to bleomycin and NCS.     

Enhancement of X-ray-induced sister chromatid exchanges in hypoxic cells

In these studies we have used wild-type Chinese hamster ovary cells (AA8) and a mutant cell line (UV-41) deficient in excision repair to compare sister chromatid exchange (SCE) induction after X irradiation under oxic and hypoxic conditions. X irradiation of AA8 cells under oxic conditions induced only a slight increase in SCEs, whereas at each dose tested a significantly greater number of SCEs were induced in hypoxic cells. When AA8 cells were X-irradiated and the addition of bromodeoxyuridine (BrdU) was delayed for 20 h to allow DNA lesions to be repaired, the levels of SCEs detected in both oxic and hypoxic cells returned to background levels. X irradiation of UV-41 cells also induced only a slight increase of SCEs in oxic cells, whereas a significant number of SCEs were induced in hypoxic cells. However, in contrast to results with AA8 cells, when hypoxic UV-41 cells were X-irradiated and the addition of BrdU was delayed for 20 h, the number of SCEs remained significantly above background levels. In combination with previous alkaline elution data, these results are consistent with the possibility that DNA-protein crosslinks are responsible for the SCEs induced by X irradiation of hypoxic cells. Irrespective of the mechanism(s) involved, the data presented suggest that the SCE assay may potentially aid in the detection of hypoxic tumor cells.     

Low doses of alpha particles do not induce sister chromatid exchanges in bystander Chinese hamster cells defective in homologous recombination

We reported previously that the homologous recombinational repair (HRR)-deficient Chinese hamster mutant cell line irs3 (deficient in the Rad51 paralog Rad51C) showed only a 50% spontaneous frequency of sister chromatid exchange (SCE) as compared to parental wild-type V79 cells. Furthermore, when irradiated with very low doses of alpha particles, SCEs were not induced in irs3 cells, as compared to a prominent bystander effect observed in V79 cells [H. Nagasawa, Y. Peng, P.F. Wilson, Y.C. Lio, D.J. Chen, J.S. Bedford, J.B. Little, Role of homologous recombination in the alpha-particle-induced bystander effect for sister chromatid exchanges and chromosomal aberrations, Radiat. Res. 164 (2005) 141-147]. In the present study, we examined additional Chinese hamster cell lines deficient in the Rad51 paralogs Rad51C, Rad51D, Xrcc2, and Xrcc3 as well as another essential HRR protein, Brca2. Spontaneous SCE frequencies in non-irradiated wild-type cell lines CHO, AA8 and V79 were 0.33SCE/chromosome, whereas two Rad51C-deficient cell lines showed only 0.16SCE/chromosome. Spontaneous SCE frequencies in cell lines defective in Rad51D, Xrcc2, Xrcc3, and Brca2 ranged from 0.23 to 0.33SCE/chromosome, 0-30% lower than wild-type cells. SCEs were induced significantly 20-50% above spontaneous levels in wild-type cells exposed to a mean dose of 1.3mGy of alpha particles (<1% of nuclei traversed by an alpha particle). However, induction of SCEs above spontaneous levels was minimal or absent after alpha-particle irradiation in all of the HRR-deficient cell lines. These data suggest that Brca2 and the Rad51 paralogs contribute to DNA damage repair processes induced in bystander cells (presumably oxidative damage repair in S-phase cells) following irradiation with very low doses of alpha particles.     

Distribution of sister chromatid exchanges on the mouse chromosomes in vivo with reference to the replication properties of the X chromosome

Frequency of sister chromatid exchanges (SCE) were recorded separately for different chromosomes from bone marrow cells of female mice of the two genetic strains (C3H/S and C57BL/6J). SCEs were evaluated following different doses of 5-bromo-2'-deoxyuridine (BrdU) as nine hourly i.p. injections. The SCE per cell increased with increasing BrdU doses which was slightly higher in C3H/S than in the C57BL/6J. SCEs per cell were variable at every treatment-strain combination, possibly reflecting the heterogeneous nature of the bone marrow cells. In general, there is a positive correlation between SCE per chromosome and the relative chromosome length. Total SCEs on one of the large chromosomes (most likely the X chromosome), however, are significantly higher than expected on the basis of relative length alone. Most of this increase is attributable to one of the homologues of this chromosome, which is not in synchrony with the rest of the chromosomes and may represent the late-replicating X. These results when viewed in the light of replication properties of the heterochromatinized X, suggest a direct involvement of DNA replication in SCE formation and may argue against the replication point as the sole site for the SCEs.     

Genotoxic effects induced by fotemustine and vinorelbine in human lymphocytes

The aim of this study was to investigate the in vitro genotoxic effects of the anticancer drugs fotemustine and vinorelbine on human lymphocytes and to determine individual and sex-related responses to these drugs. Fotemustine is a DNA-alkylating drug while vinorelbine is a semi-synthetic Vinca alkaloid. The study was carried out with twenty independent healthy donors for each drug. We have tested the ability of these drugs to induce chromosome aberrations (CAs) and sister chromatid exchanges (SCEs) as well as effect on the mitotic index (MI) in cultured human lymphocytes. Fotemustine was shown to induce CAs and SCEs at all concentrations tested (2, 4 and 8 microg/ml) in a dose-dependent manner. Additionally it also decreased the mitotic index in a similar dose-dependent manner. Vinorelbine had no effect on structural CAs, but it significantly increased the numerical CAs at all doses tested (0.5, 1 and 2 microg/ml). Vinorelbine also induced SCE events and increased the MI values. Two-way analyses of variance were used to compare the individual and gender-related susceptibilities to fotemustine and vinorelbine with respect to the CA, SCE and MI values. The results indicated that individuals in fotemustine treatment groups showed different genotoxic responses with respect to CA and SCE induction and additional findings indicated a gender-specific response in this group. Individuals in the vinorelbine test group also exhibited statistically significant numerical CA, SCE and MI responses to vinorelbine. A statistically significant gender-related SCE response to this drug was also evident. This study indicates that these drugs have potentially harmful effects on human health.     

Levels of sister-chromatid exchanges in hybrids between Bloom syndrome B-lymphoblastoid cells and various cell lines with lymphoid malignancy

The present study has been undertaken to examine the effect of cell hybridization of Bloom syndrome (BS) B-lymphoblastoid cell lines (LCLs) and various cell lines from lymphoid malignancies in order to clarify the relationship between sister-chromatoid exchange (SCE) and malignant conditions. Cell hybridization studies have shown that though BS high-SCE frequencies were completed by fusion with normal cells, fusion with various malignant cell lines did not result in complete normalization of BS SCEs, with 15-30 SCEs remaining per hybrid cell, demonstrating possibly common defects in DNA of BS and malignant cells. These findings strongly support the idea that the characteristic high SCE frequency in BS cells has some connection with the malignant condition, and that at least one step in carcinogenesis is either accompanied by the production of SCEs, or that SCEs themselves cause such a step to occur.     

Cell-stage dependence of mutagen-induced sister-chromatid exchanges in human lymphocyte cultures

The induction of sister-chromatid exchanges (SCEs) was studied in phytohemagglutinin (PHA)-stimulated human lymphocytes exposed for 1 h to mitomycin C (MMC, 3 X 10(-6) M), ethyl methanesulphonate (EMS, 2 X 10(-2) M), or 4-nitroquinoline-1-oxide (4NQO, 3 X 10(-5) M) at various cell-cycle stages of 72-h cultures. The doses of the chemical were chosen to give about 20 SCEs per cell when treated at Go. The SCE frequency increased almost linearly with MMC or EMS treatments at later times after PHA stimulation, peaking with those at 36 h (at around the first G1/S boundary in the 2 consecutive cell cycles, which was revealed by concomitant experiments), and then decreased with subsequent treatment times. Cell-cycle kinetics and the cell stages at which the cells were treated were measured by autoradiography and sister-chromatid differential staining. The data show that MMC and EMS produce larger numbers of SCEs when treated at stages closer to the beginning of S, and that the most efficient time of treatment is the G1/S boundary in the first cell cycle of the two consecutive cycles before sampling. Pulse treatment with EMS caused about 3 times larger inductions of SCEs when done at late G1/early S(G1/S boundary) in the first cell cycle compared to that at G0/early G1, whereas identical exposure to MMC at the first G1/S boundary produced only 1.5 times larger numbers of SCEs than that at G0/early G1. EMS and MMC both, however, induced 30-40% larger numbers of SCEs when treated at the G1/S boundary in the first cell cycle than when treated at the second cell cycle before sampling. On the contrary, treatment with 4NQO led to the induction of about the same numbers of SCEs even when treated at different cell-cycle stages before the second G1/S boundary. The SCE frequency in 4NQO-treated cells then decreased with subsequent treatment times.     

Exposure of nuclear medicine patients to ionizing radiation is associated with rises in HPRT- mutant frequency in peripheral T-lymphocytes

When Chinese hamster fibroblasts were exposed to hydrogen peroxide or to a system consisting of xanthine oxidase and hypoxanthine, which generates superoxide anion plus hydrogen peroxide, sister-chromatid exchanges (SCEs) were formed in a dose-dependent manner. When the iron-complexing agent o-phenanthroline was present in the medium, however, the production of these SCEs was completely inhibited. This fact indicates that the Fenton reaction: Fe2+ + H2O2----OH0 + OH- + Fe3+ is responsible for the production of SCEs. When O2- and H2O2 were generated inside the cell by incubation with menadione, the production of SCE was prevented by co-incubation with copper diisopropylsalicylate, a superoxide dismutase mimetic agent. The most likely role of O2- is as a reducing agent of Fe3+: O2- + Fe3+----Fe2+ + O2, so that the sum of this and the Fenton reaction, i.e., the iron-catalyzed Haber-Weiss reaction, provides an explanation for the active oxygen species-induced SCE: H2O2 + O2(-)----OH- + OH0 + O2. According to this view, the OH radical thus produced is the agent which ultimately causes SCE. These results are discussed in comparison with other mechanisms previously proposed for induction of SCE by active oxygen species.     

Aphidicolin-Resistant Mutants of Mouse Lymphoma L5178y Cells with a High Incidence of Spontaneous Sister Chromatid Exchanges

Two aphidicolin-resistant cell mutants (AC 12 and AC 41) with a fourfold increase in spontaneous frequency of sister chromatid exchanges (SCEs) were obtained out of over 400 aphidicolin-resistant mutants isolated from mouse lymphoma L5178Y cells. They also exhibited three- to fourfold increases in spontaneous frequency of chromosome aberrations (CAs). To determine whether the high level of SCE frequency in AC 12 is caused by 5-bromodeoxyuridine (BrdUrd) used for visualizing SCEs, the effect of BrdUrd incorporated into DNA on SCE induction was analyzed. The SCE frequencies in AC 12 remained constant at BrdUrd incorporation levels corresponding to 2-90% substitution for thymidine in DNA. In addition, the small amount of BrdUrd incorporated into both daughter and parenteral DNA strands in AC 12 had minimal effect on SCE induction. Furthermore, AC 12 and AC 41 were slightly resistant to BrdUrd with respect to the induction of CAs, the inhibition of cell-cycle progression and the decrease in mitotic activity. These findings suggest that the high incidence of SCEs in AC 12 and AC 41 is formed by their intrinsic defects, not by the effects of BrdUrd used. The analysis of SCE frequencies in hybrid cells between these mutants and the parental L5178Y revealed that the genetic defects in AC 12 and AC 41 appear to be recessive, and that these two mutants belong to the same complementation group. Furthermore, AC 12 belonged to a different complementation group from ES 4, which was isolated previously from L5178Y as an SCE mutant with a twofold higher frequency of spontaneous SCEs. This finding indicates that at least two different genetic defects participate in the formation of the high incidence of spontaneous SCEs in mouse cells. These SCE mutants would provide valuable cell materials for studying the molecular mechanism of SCE formation.     

Mitomycin-C-induced sister-chromatid exchanges and cell-cycle kinetics in lymphocytes from patients with Klinefelter syndrome

The chromosomal sensitivity to mitomycin-C (MMC) and cell-cycle kinetics in cells from patients with Klinefelter syndrome, a sex chromosomal disorder giving a high risk of malignant tumor, were studied by techniques of sister-chromatid exchanges (SCEs). The frequencies of MMC-induced SCEs increased in proportion to the increase in MMC concentration in both patient and normal control cells. At low levels of MMC there were no significant differences in SCE frequencies between the patient and normal control cells, but at MMC concentrations of 3 X 10(-8) M (p less than 0.05) and 1 X 10(-7) M (p less than 0.01), significant increases in the frequency of MMC-induced SCEs were observed in cells from patients compared to cells from normal controls. Although the analysis of cell-cycle kinetics both after various culture times and after treatment with MMC revealed that there were no significant differences between the patient and normal control cells, patients with Klinefelter syndrome showed a tendency to cell-cycle delays after treatment with MMC in comparison with normal controls.     

Sister chromatid exchanges in the human active and inactive X chromosomes

Four human female fibroblast strains with an i(Xq) or derivative X chromosome as a cytological marker for the inactive X chromosome were used to determine the frequency of sister chromatid exchanges (SCEs) in the active and inactive X chromosomes. No significant difference in SCE frequency between the active and inactive X chromosomes was observed. Therefore, the state of chromatin condensation and the late DNA replication in the facultative heterochromatin of the inactive X chromosome do not appear to influence the SCE frequency.     

Disparate effects of 5-bromodeoxyuridine on sister-chromatid exchanges and chromosomal aberrations in Bloom syndrome fibroblasts

The existence of a high frequency of spontaneous sister-chromatid exchanges (SCEs) in Bloom syndrome (BS) has thus far been supported by data on a small number of BS cell lines. To examine the cause of baseline SCEs more broadly, the frequencies of SCEs, as well as chromosomal aberrations (CAs) in 4 additional BS fibroblast strains were compared, under different assay and cell culture conditions, with those of normal cells in the range of approximately 0.9-90% 5-bromodeoxyuridine (BrdUrd) substitution into template DNA. SCEs at low levels of BrdUrd substitution were detected by an extremely sensitive immunofluorescent technique. From approximately 0.9% to 4.5% BrdUrd substitution, the SCE frequency in BS cells remained constant, at a level (40/cell) 8 times higher than that of normal cells. As BrdUrd substitution increased further, the SCE frequency in BS cells increased almost linearly, reaching 70-100 per cell at approximately 90% substitution, while the SCE increment in control fibroblasts was less than 5 per cell. Analysis of SCEs in 3 successive replication cycles similarly revealed that the SCE increment in BS cells depended on BrdUrd only at a high BrdUrd substitution level. In contrast to data on SCEs, CA induction by incorporated BrdUrd in BS cells was only slightly higher than that in normal cells. Thus, BS cells are extremely sensitive to BrdUrd for SCE induction, but much less so for CA induction.     

Sister-chromatid exchange in childhood in relation to age and sex

Small children have been found to have a lower SCE/cell than adults and in recent reports females have had higher SCEs/cell than males. We here describe the relationship between SCE/cell and age and sex in 46 girls and 39 boys with an age range of 1.4-19.2 years and 2.6-18.7 years, respectively. For the calculation a transformation y = (sum SCE)1/2 + (sum SCE + 1)1/2 was used. The best fit to our material was represented by the equation y = b0 + b1 X log age. A common slope (b1) could be used for the boys and girls. This slope was significantly different from zero (P less than 0.0005). The levels of the regression lines for the two sexes were different (P = 0.0006). The girls had a 0.55-0.7 higher SCE/cell than the boys, depending on age. The following equations were found: Girls: y = 22.49 + 6.53 X log age. Boys: y = 21.11 + 6.53 X log age. By this model 43% of the variation in y could be explained. As a consequence of the result it is absolutely essential, when planning studies of children, to use age-matched groups to decrease the variability of the test system.     

A comparison of sister-chromatid exchange in mouse peripheral blood lymphocytes exposed in vitro and in vivo to phosphoramide mustard and 4-hydroxycyclophosphamide

Cyclophosphamide (CP) and two of its known metabolites, 4-hydroxycyclophosphamide (4-OHCP) and phosphoramide mustard (PAM), were analyzed for their ability to induce sister-chromatid exchanges (SCEs) in mouse peripheral blood lymphocytes (PBLs) in vitro and in vivo. At equimolar concentrations, CP is a more potent SCE inducer in vivo than PAM and PAM and 4-OHCP induce equal numbers of SCEs in a dose-dependent manner. The present study also shows that these metabolites of CP are more potent SCE inducers than CP itself in vitro. This relationship might be explained by the differences in pharmacokinetics of these compounds.     

Increased sister chromatid exchange events in the human late replicating X

Summary Human female blood cultures were labeled with BrdU for detecting sister chromatid exchanges (SCEs) by the Hoechst 33258 fluorescence technique. Late labeling with ³H-thymidine and autoradiography allowed the identification of the late replicating X. The mean number of SCEs in the cells was 13. The isopycnotic X showed an exchange frequency according to its relative length in the karyotype; in the late replicating X a doubled number of SCE events was observed.     

Sister Chromatid Exchanges Are Mediated by Homologous Recombination in Vertebrate Cells

Sister chromatid exchange (SCE) frequency is a commonly used index of chromosomal stability in response to environmental or genetic mutagens. However, the mechanism generating cytologically detectable SCEs and, therefore, their prognostic value for chromosomal stability in mitotic cells remain unclear. We examined the role of the highly conserved homologous recombination (HR) pathway in SCE by measuring SCE levels in HR-defective vertebrate cells. Spontaneous and mitomycin C-induced SCE levels were significantly reduced for chicken DT40 B cells lacking the key HR genes RAD51 and RAD54 but not for nonhomologous DNA end-joining (NHEJ)-defective KU70(-/-) cells. As measured by targeted integration efficiency, reconstitution of HR activity by expression of a human RAD51 transgene restored SCE levels to normal, confirming that HR is the mechanism responsible for SCE. Our findings show that HR uses the nascent sister chromatid to repair potentially lethal DNA lesions accompanying replication, which might explain the lethality or tumorigenic potential associated with defects in HR or HR-associated proteins.