SCE evaluations in human lymphocytes after G0 exposure to mitomycin C Lack of expression of MMC-induced SCEs in cells that have undergone greater than two in vitro divisions

We conducted a series of experiments designed to determine whether DNA damage induced in G₀ lymphocytes by mitomycin C (MMC) would be expressed as sister-chromatid exchanges during the second and third post-treatment cell cycles. Lymphocytes from normal donors were exposed to MMC for 2 h prior to culture in the presence of phytohemagglutinin. MMC-treated and control cells were subsequently exposed to bromodeoxyuridine (BrdUrd) for the entire culture period (i.e. 48 h or 72 h) or for the terminal 24 h of 72-h cultures. We observed a 3–4-fold increase in SCEs in MII metaphases from lymphocytes treated with MMC and cultured in the presence of BrdUrd for the entire culture period. In contrast, in replicate cultures of MMC-treated lymphocytes that were exposed to BrdUrd for the terminal 24 h only, the SCE frequency in uniformly harlequinized metaphases was not significantly different from that observed in control cultures. We interpret these data as providing evidence that MMC-induced lesions (or alterations) in the DNA of G₀ lymphocytes are probably expressed as SCEs during the first period of mitogeninduced DNA synthesis, and that these lesions do not persist and give rise to SCEs in subsequent cell divisions.     

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

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

Cytogenetic effects of penicillamine

Penicillamine (PA), a drug used for the treatment of rheumatoid arthritis induces sister-chromatid exchanges (SCEs) and chromosome aberrations in cultivated mammalian cells. PA in concentrations from 400 micrograms/ml upward induced SCEs and proliferative delay in human blood cultures when added for the last 24 h of the culture period. In V79 Chinese hamster cells SCE induction was found after acute exposure to PA before the addition of BrdUrd and after chronic exposure during one cell cycle in the presence of BrdUrd. The effect of PA on SCE frequencies occurred both after treatment in complete medium and in serum-free medium and was not influenced by the application of an S9 mix. The simultaneous addition of peroxidase reduced the PA-induced SCEs whereas catalase did not show any effect. Chromosome analysis in the first mitosis after PA treatment revealed a significant increase in the incidence of chromosome aberrations and endoreduplication. The results are discussed with respect to the cause and the significance of the observed effects in connection with mutagenicity testing.     

Presence of abnormally high incidences of sister chromatid exchanges in three successive cell cycles in Bloom's syndrome lymphocytes

The frequencies of sister chromatid exchanges (SCEs) were examined in phytohaemagglutinin-stimulated blood lymphocytes of a normal individual, a Bloom's syndrome heterozygote (bl/+), and two Bloom's syndrome homozygotes (bl/bl). To determine the baseline SCE frequencies, lymphocytes were cultured with various concentrations of 5-bromodeoxyuridine (BrdUrd) for two cell cycles. The incidence of SCEs per two cell cycles inbl/bl lymphocytes levelled off at BrdUrd concentrations below 10 g/ml while that in normal andbl/+ lymphocytes stayed constant below 7.5 g/ml. The baseline SCE frequency in bl/bl cells was ten times higher than that in normal andbl/+ cells. At BrdUrd concentrations above 15 g/ml, SCEs inbl/bl cells were induced more frequently than in normal andbl/+ cells. These results indicate that at low concentrations BrdUrd has a minimal effect on the induction of SCEs in all individuals, while at higher concentrations the BrdUrd incorporated inbl/bl cells has a larger effect than that in normal andbl/+ cells. To elucidate the effect of BrdUrd incorporated into the daughter and parental DNA strands on SCE induction, SCEs occurring during each cell cycle were examined separately in three-way or two-way differentially stained, third-cycle metaphases. The incidence of SCEs detected in each cell cycle at 5 g/ml BrdUrd was constant in all individuals and the rates of SCEs in each cell cycle inbl/bl cells were remarkably higher than those observed in normal andbl/+ cells. These findings strongly indicate that most of the abnormally increased SCEs in thebl/bl cells used in our study occurred independently of any effect of BrdUrd incorporated into both the daughter and parental DNA strands. In addition, an abnormal response ofbl/bl cells to BrdUrd was not found for cell cycle progression or chromosomal aberration induction. Thus, the bl/bl cells did not exhibit an abnormal hypersensitivity to BrdUrd. From these results, it seems quite probable that the abnormally increased SCEs in thebl/bl lymphocytes used here were spontaneous.     

Effect of exogenous thymidine on sister-chromatid exchange frequency in Chinese hamster ovary cells with bromodeoxyuridine- and chlorodeoxyuridine-substituted chromosomes

There are conflicting reports on the effect of exogenous thymidine (dThd) on the frequency of sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. Thymidine has been reported either to increase or to have no effect on SCE frequency under similar experimental conditions. To resolve this controversy, we have carried out a series of experiments to examine the effect of dThd on CHO cells cultured with 5-bromodeoxyuridine (BrdUrd). In addition, we have examined the effect of dThd on CHO cells cultured with 5-chlorodeoxyuridine (CldUrd), a much more potent inducer of SCEs than BrdUrd. The addition of 100 microM dThd to the culture medium caused a consistent decrease in the yield of SCEs in cells grown in BrdUrd for two cell cycles. The decrease was even greater when cells were grown in dThd and CldUrd. Analysis of twin and single SCEs indicated that dThd must be present during the first cell cycle to reduce the frequency of SCEs. Because excess dThd is thought to have an effect when DNA replicates on a template substituted with a halogenated nucleoside, dThd at concentrations from 100 microM to 9 mM was added to cultures for the second cell cycle after a first cell cycle in BrdUrd. In this experiment, the presence of dThd increased SCE frequency in a dose-dependent manner. The results suggest that if dThd competes with halogenated nucleosides and thus decreases their incorporation into DNA, SCEs are suppressed in the subsequent cell cycle, whereas if excess dThd creates a dNTP pool imbalance, SCEs can be increased.     

Both cross-links and monoadducts induced in DNA by psoralens can lead to sister chromatid exchange formation

The relative importance of DNA-DNA cross-links and bulky monoadducts in sister chromatid exchange (SCE) formation was investigated in three human fibroblast cell lines with different repair capabilities. These cell lines included normal cells, which can repair both classes of lesions; xeroderma pigmentosum (XP) cells, which cannot repair either psoralen-induced cross-links or monoadducts; and an XP revertant that repairs only cross-links and not monoadducts. SCEs were induced by two psoralen derivatives, 4'-hydroxymethyl-4,5',8-trimethylpsoralen (HMT) and 5-methylisopsoralen (5-MIP). After activation with long-wave ultraviolet light, HMT produces cross-links and monoadducts in DNA, whereas 5-MIP produces only monoadducts. In normal human cells both psoralens induced SCEs, but if cells were allowed to repair for 18 h before bromodeoxyuridine (BrdUrd) was added for SCE analysis, the SCE frequency was significantly reduced. XP cells showed an SCE frequency that remained high regardless of whether SCEs were analyzed immediately after psoralen exposure or 18 h later. In the XP revertant that repairs only cross-links, both psoralens induced a high yield of SCEs when BrdUrd was added immediately after psoralen treatment. When XP revertant cells were allowed 18 h to repair before addition of BrdUrd, the SCEs induced by HMT were greatly reduced, whereas those induced by 5-MIP were only slightly reduced. These observations indicate that both cross-links and monoadducts are lesions in DNA that can lead to SCE formation.     

Sister-chromatid exchanges and cell-cycle kinetics in human lymphocyte cultures exposed to alkylating mutagens: apparent deformity in dose-response relationships

Experiments have been carried out using human whole-blood cultures to determine the effects of sampling times and of the duration of 5-bromodeoxyuridine (BrdUrd) treatment before fixation on sister-chromatid exchange (SCE) frequencies following exposure to mitomycin C (MMC). Cells were pulse treated for 1 h with 3 X 10(-6) M MMC at G1, and then sampled at 4-h intervals up to 88 h after stimulation of cultures with phytohemagglutinin (PHA). Results showed that this MMC treatment induced a 5-6 h proliferation delay per cell cycle, and that SCE frequencies first increased with time of fixation, peaking at 68 h, and then decreased. When cells were similarly treated with MMC, but subsequently exposed to BrdUrd for various times before fixation of cultures at 72 h, the SCE frequencies markedly increased with increasing durations of BrdUrd incubation times. These data indicate that, in mutagen-treated cultures, lymphocytes having relatively longer cell-cycle times show a higher mean frequency of SCEs. In a subsequent experiment, cells were treated for 1 h with increasing doses of MMC or 4-nitroquinoline 1-oxide (4NQO) at 0, 24, or 48 h, and then fixed at 72 h after PHA stimulation. Results showed that the optimal treatment times at which the agents could most efficiently produce SCEs were different for MMC and 4NQO, and that the dose-response curves tended to 'bend down' at very high doses; that is, treatments with very high doses induced smaller than expected numbers of SCEs. However, cells similarly treated with very high doses showed a higher, expected frequency of SCEs when sampled at 84 h, but again had a lower than expected SCE frequency when fixed at 96 h. The results indicate that there is an optimal time for sampling at which one can observe the maximum increase in SCE frequencies following mutagen exposure, and strongly suggest that the higher the dose, the later the optimal sampling time. Because of the apparent deformity of dose-response curves obtained after various treatments and sampling times, it seems necessary that extra fixation-time points be included in test protocols so as to avoid false negatives or confirm possible positives.     

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.     

Contribution of incorporated 5-bromodeoxyuridine in DNA to the frequencies of sister-chromatid exchanges induced by inhibitors of poly-(ADP-ribose)-polymerase

3-Aminobenzamide and benzamide, two potent inhibitors of poly-(ADP-ribose)-polymerase increase the frequencies of SCEs in Chinese hamster ovary cells in a dose-dependent manner. SCEs were studied in cells in which the inhibitors were present either during the first cell cycle or the second cell cycle or both. Most of the induced SCEs were found to be formed during the second cell cycle in which BU-containing DNA was used as template for DNA synthesis. In cells which were pregrown for 4 cell cycles in the presence of BrdUrd, in order to obtain both sister chromatids bifiliarly substituted with BU in their DNA, it was found that the presence of inhibitor even in the first cell cycle increased the frequencies of SCEs. It is concluded that the incorporated BrdUrd plays an important role in the origin of spontaneous and induced SCEs. 3-Aminobenzamide alone or benzamide in the presence of BrdUrd during culture, did not increase the frequencies of mutations to HGPRT^? in these cells.     

Bimodal induction of sister-chromatid exchanges by luminol,an inhibitor of poly(ADP-ribose) synthetase,during the S-phase of the cell cycle

The cell cycle dependence of sister chromatid exchanges (SCEs) induced by luminol, a new potent inhibitor of poly(ADP-ribose) synthetase, was studied in Chinese hamster V79 cells. Continuous treatment with luminol during two whole cell cycles in the presence of 5-bromo-2-deoxyuridine (BrdUrd), or in the first or second cycle induced SCEs very efficiently in a linear dosedependent manner. However, no enhancement of SCE levels was observed after luminol treatment in a cycle preceding BrdUrd treatment, in contrast to results found with other strong SCE inducers such ascis-diammine-dichloroplatinum (II) (CDDP) and mitomycin C (MMC). Luminol was about ten times as effective in inducing SCEs as 3-aminobenzamide (3AB), an inhibitor of the NAD⁺ site of poly(ADP-ribose) synthetase. The induction of SCEs by luminol was restricted to the Sphase of the cell cycle with peaks at an early and a late stage, corresponding to the biphasic replication of DNA. The mechanism of SCE appears to be the same at the early and late stages of S-phase for luminol-induced SCE formation.     

Physical,chemical, and biological factors affecting sister-chromatid exchange induction in human lymphocytes exposed to mitomycin C prior to culture

In experiments to assess the effects of several biological, chemical, and physical variables on sister-chromatid exchange (SCE) induction in cultured lymphocytes exposed to mitomycin C (MMC) before PHA stimulation we observed: (1) high SCE frequencies in female cells, and normal SCE frequencies in Y-bearing metaphases in mixed cultures containing equal numbers of MMC-treated female lymphocytes and untreated male lymphocytes; (2) small, but statistically significant, decreases in SCEs with increasing pH after G₀ exposure in the pH range 6.6–7.6; (3) pronounced reductions in MMC-induced SCEs in lymphocytes exposed at 4°C vs. 37°C. In other studies, SCE induction was evaluated in cultures exposed during G₀ to MMC concentrations ranging from 0.25 to 2.5 μg/ml for varying time intervals ranging from 5 min to 24 h. For all concentrations tested SCE induction varied as a linear function of G₀ exposure time. To compare SCE induction between cultures, we calculated the mean frequencies of SCEs induced per metaphase/unit dose MMC/unit G₀ exposure time (SCE/μg/h). A mean frequency of 20.7 ± 4.8 SCE/μg/h was observed for 41 lymphocyte cultures suggesting that a single term adequately describes the rate of SCE induction following G₀ exposure to a 10-fold range in concentration of MMC for time intervals of 30 min to 24 h.     

Analyses for in vitro growth conditions,cytokinetics, and sister chromatid exchanges in lymphocytes cultured from the Sprague-Dawley rat

Summary Peripheral blood samples from Sprague-Dawley rats gave successful lymphocyte growth in GIBCO: IA, RPMI 1640, and Eagle's minimum essential medium (MEM) culture media. Various growth conditions, cytokinetics, and sister chromatic exchange (SCE) induction were studied using reconstituted GIBCO 1A only. Neither methoxyflurane anesthesia of the rats before sampling nor washing of the cells with phosphate buffered saline affected the mitotic index. Cultures treated with [³H]thymidine showed the lymphocytes entering into DNA synthesis after approximately 24 h. The time at which BUdR (5-bromo-2′ deoxyuridine) was added, i.e. 0 vs. 24 h incubation, had minimal effect on the mitotic index of cultures harvested at 48 h. However, when harvest was extended to 72 h, mitotic activity was greater in the cultures treated with BUdR at 24 h. No significant differences in mitotic index and the number of average lymphocyte division were detected in cultures exposed to 0.3 to 0.5 μg/ml BUdR at 24 h and harvested at 72 h. Although SCE frequencies increased in the presence of BUdR, the baseline level of SCEs was estimated to be 5 to 6/cell. Average generation time of the lymphocytes dividing between 48 and 72 h was 16.5 h. Because of its simplicity of culture and the reproducible nature of its in vitro growth kinetics, the Sprague-Dawley rat lymphocyte is a suitable model for cytogenetic investigations.     

Analysis of mutagenesis and sister-chromatid exchanges induced by 5-bromo-2'-deoxyuridine in somatic hybrids derived from Syrian hamster melanoma cells and Chinese hamster ovary cells

Somatic cell hybrids were derived from the fusion of Chinese hamster ovary (CHO) cells and Syrian hamster melanoma cells (2E). These two cell lines had previously been shown to differ in their response to the induction of mutations and sister-chromatid exchanges (SCEs) by 5-bromo-2'-deoxyuridine (BrdUrd) (Kaufman, 1987). The parental cells and a number of representative, independent hybrid clones were tested for their response to both the INC and REP mutagenesis protocols. INC mutagenesis involves the incorporation of BrdUrd into DNA under conditions of deoxyribonucleoside triphosphate (dNTP) pool imbalance, while REP mutagenesis involves the replication of 5-bromouracil-substituted DNA in the presence of dNTP pool imbalance. When tested for the toxic effects of high concentrations of BrdUrd and for the induction of mutations by the INC protocol, the hybrid clones all expressed the 2E phenotype, i.e., sensitivity to relatively low concentrations of BrdUrd and thymidine for the induction of mutations, dNTP pool perturbation, and the toxic effects of BrdUrd. When the hybrid clones were tested for the induction of mutations and SCEs by the REP protocol, it was found that they expressed the 2E phenotype for the induction of mutations and the CHO phenotype for the induction of SCEs. Thus, various aspects of the 2E phenotype, such as high mutation frequencies associated with large dNTP pool perturbations, appeared to be dominantly expressed in the cell hybrids, while the lack of induction of SCEs by these mutagenic conditions in 2E cells was found to be a recessive characteristic.     

Analysis of mutagenesis and sister-chromatid exchanges induced by 5-bromo-2′-deoxyuridine in somatic hybrids derived from Syrian hamster melanoma cells and Chinese hamster ovary cells

Somatic cell hybrids were derived from the fusion of Chinese hamster ovary (CHO) cells and Syrian hamster melanoma cells (2E). These two cell lines had previously been shown to differ in their response to the induction of mutations and sister-chromatid exchanges (SCEs) by 5-bromo-2′-deoxyuridine (BrdUrd) (Kaufman, 1987). The parental cells and a number of representative, independent hybrid clones were tested for their response to both the INC and REP mutagenesis protocols. INC mutagenesis involves the incorporation of BrdUrd into DNA under conditions of deoxyribonucleoside triphosphate (dNTP) pool imbalance, while REP mutagenesis involves the replication of 5-bromouracil-substituted DNA in the presence of dNTP pool imbalance. When tested for the toxic effects of high concentrations of BrdUrd and for the induction of mutations by the INC protocol, the hybrid clones all expressed the 2E phenotype, i.e., sensitivity to relatively low concentrations of BrdUrd and thymidine for the induction of mutations, dNTP pool perturbation, and the toxic effects of BrdUrd. When the hybrid clones were tested for the induction of mutations and SCEs by the REP protocol, it was found that they expressed the 2E phenotype for the induction of mutations and the CHO phenotype for the induction of SCEs. Thus, various aspects of the 2E phenotype, such as high mutation frequencies associated with large dNTP pool perturbations, appeared to be dominantly expressed in the cell hybrids, while the lack of induction of SCEs by these mutagenic conditions in 2E cells was found to be a recessive characteristic.     

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

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

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.     

Alterations in the baseline sister-chromatid exchange frequency in human lymphocyte culture following a number of cell divisions

A significant decrease in the baseline of sister-chromatid exchanges (SCEs) was observed in cultured human lymphocytes, if 5-bromodeoxyuridine (BrdU) was added after 60 h of culture, and the cells were harvested at least 24–30 h after BrdU exposure. This decrease is supposed to occur if at least one cell division takes place before the addition of BrdU. For cytogenetic monitoring of mutagenic environmental factors, using human lymphocyte cultures, it is assumed that two time periods are sufficient for comparison.     

Induction of sister-chromatid exchanges by the replication of 5-bromouracil-substituted DNA under conditions of nucleotide-pool imbalance

The induction of sister-chromatid exchanges (SCEs) by the replication of 5-bromouracil(BrUra)-containing DNA under conditions of nucleotide-pool imbalance was investigated. A modification of a protocol developed for the induction of mutations under these conditions (E.R. Kaufman, Mol. Cell. Biol., 4, 2449-2454, 1984) was used. To induce SCEs, Chinese hamster ovary cells were grown under non-mutagenic conditions which allowed the uniform incorporation of BrUra into their DNA at specific levels of substitution for thymine residues (25, 50 and 75% BrUra substitution). After 4 and 5 days of growth, the cells, which had incorporated BrUra into their DNA, were washed free of 5-bromodeoxyuridine (BrdUrd) and provided with fresh culture medium supplemented with various concentrations of thymidine (10 microM to 3 mM) and no BrdUrd. The cells were allowed to replicate their BrUra-containing DNA under these conditions, in the absence of BrdUrd, for two rounds of DNA synthesis to achieve sister-chromatid differentiation, and second-division metaphases were scored for SCEs. The results of these studies indicated that the SCEs observed were proportional to the level of BrUra substituted for thymine in the cellular DNA, were induced by increasing concentrations of thymidine in the culture medium during replication of the BrUra-containing DNA, correlated well with the induction of mutations to thioguanine resistance and to ouabain resistance, correlated with increases in the intracellular levels of dTTP and dGTP generated by the high concentrations of thymidine. These findings provide direct evidence for the induction of SCEs by the replication of BrUra-containing DNA and for the importance of the pools of nucleoside triphosphate precursors for DNA replication in these processes. When the effects of 3-aminobenzamide, a potent inhibitor of poly(ADP-?ibose) synthesis, were tested, it was found that 3-aminobenzamide significantly increased SCEs, but it had no effect on mutations induced.     

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

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