Function and organization of the G region of bacteriophage Mu; Host specificity determined by gene splicing

Chinese hamster ovary (CHO) cells were exposed to [³H]ethyl nitrosourea (ENU) or [³H]ethyl methanesulfonate (EMS) and the following DNA ethylation products were quantitated: 3- and 7-ethyladenine, O²-ethylcytosine, 3-, 7- and O⁶-ethylguanine, O²- and O⁴-ethyldeoxythymidine and the representative ethylated phosphodiester, deoxythymidylyl (3′–5′)ethyl-deoxythymidine. When mutations at the hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus induced by these same treaments were compared with the observed ethylation products, mutations were found to correlate best with 3- and O⁶-ethylguanine. EMS induced approximately twice as many sister-chromatid exchanges (SCEs) as ENU at doses yielding equal mutation frequencies. When SCEs were indirectly compared with DNA ethylation products, 3-ethyladenine and ethylated phosphodiesters related best to SCE formation. Because mutation and SCE induction appear, at least in part, to be related to different DNA adducts, SCE induction by simple ethylating agents may not be a quantitative indicator of potentially mutagenic DNA damage.     

Inhibition of protein synthesis antagonizes induction of sister-chromatid exchanges by exogenous agents

Cycloheximide (CH) and puromycin (PM) strongly antagonize induction of sister-chromatid exchanges (SCEs) by exogenous agents regardless of the mechanisms for initiating damage. 5-Bromodeoxyuridine (BUdR) substitution was used to monitor SCEs, but the background level of BUdR-induced SCEs was unaffected by the presence of protein inhibitors. Antagonism between DNA-damaging agents and protein inhibitors was strongest in euchromatic regions. Possible relationships between SCE formation and the mechanism of antagonism by protein inhibitors are discussed.     

DNA crosslinking, sister-chromatid exchange and specific-locus mutations

Chinese hamster ovary cells were treated with the DNA-crosslinking chemicals, mitomycin C (MMC) and porfiromycin (POR), and their monofunctional derivative decarbamoyl mitomycin C (DCMMC). After exposure, the cells were studied for the induction of sister-chromatid exchanges (SCEs) and mutations at the hypoxanthine phosphoribosyltransferase and adenine phosphoribosyltransferase loci. The frequency of SCEs varied significantly in successive sampling intervals, requiring the weighting of each interval by the percentage of second-division mitosis in that interval to obtain the mean SCE frequency for each dose. All 3 compounds were potent inducers of SCEs but weakly mutagenic. All 3 chemicals by concentration were approximately equally effective in inducing SCEs or mutations. When the induced SCEs and mutations were compared at equal levels of survival, DCMMC was slightly more effective than MMC or POR in inducing SCEs and somewhat less mutagenic. These results indicate that the DNA interstrand crosslink is not the major lesion responsible for the induction of SCE or mutation by these compounds.     

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.     

Cyclophosphamide-Induced IN VIVO Sister Chromatid Exchanges (Sce) in MUS MUSCULUS. III. Quantitative Genetic Analysis

In vivo cyclophosphamide (CP)-induced sister chromatid exchanges (SCEs) were evaluated in females from five genetic strains of mice (C57BL/6J, C3H/S, 129/ReJ, BALB/c and DBA/2) and their F₁ hybrids. Baseline (noninduced) SCE values differ significantly among strains, 129/ReJ having the lowest and DBA/2 having the highest mean SCE per cell values. In general, the baseline SCE of a given F₁ is within the range of its corresponding parental strains or near the lower parental value. Furthermore, there is a genotype-dependent increase in mean SCEs per cell with CP dose. Strain differences in SCE induction are noted particularly at the two higher CP doses (4.50 and 45.0 mg/kg). In general, F₁ hybrids involving a strain with high induced SCEs and a strain with low induced SCEs exhibit mean SCE values that are closer to the value of the lower strain. F₁ s involving two strains with high SCEs or two strains with low SCEs yield SCEs not different from parental strains. The method of diallel cross analysis showed the order of dominance of these strains in SCE induction to be 129/ReJ BALB/c C3H/S DBA/2 C57BL/6J. These results support the involvement of predominantly nonadditive genetic factors as major gene(s) in SCE induction. In addition, involvement of random and independent events in SCE induction is suggested by the distribution of SCEs which follows a Poisson distribution.     

Plant extracts induce chromosome aberrations and sister-chromatid exchanges in Chinese hamster ovary cells and human lymphocytes

Effects of extracts from Vicia faba were compared with those of Zea mays for the induction of sister-chromatid exchanges (SCEs) and of chromosome aberrations (CAs) in Chinese hamster ovary (CHO) cells. CA induction by the maize extract was also tested in human lymphocytes. The extracts from roots and leaves of Vicia faba induced CAs and SCEs in CHO cells. The extracts from maize leaves also induced SCEs and CAs in CHO cells, and CAs in human lymphocytes. Maize extracts were more potent in inducing SCEs than Vicia extracts and the SCE- and CA-inducing capacity of maize extracts decreased during preincubation before addition to cells.     

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.     

Sister-chromatid exchanges (SCEs), cell survival and mutation in HeLa s3 cells with different sensitivity to alkylating agents; evidence that SCE induction and cell survival or mutation induction are dissociable

We previously isolated N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-resistant cells, MR from HeLa S3 Mer- cells. In the present study, we have isolated 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea (ACNU)-resistant cells, ACr. The MR cells had only a little O6-methylguanine-DNA methyltransferase (MT) activity, while the ACr cells had increased MT activity and also became resistant to the cytotoxic effect of MNNG. We compared the induction of sister-chromatid exchanges (SCEs), cell survival and mutation in these HeLa S3 cells with different sensitivity to MNNG. The ACr cells were much more resistant than the parental HeLa S3 Mer- cells to cytotoxicity, mutagenicity and SCE induction by MNNG, showing a positive correlation between SCE induction and cell killing or mutation. In contrast, this positive relationship was not observed between HeLa S3 Mer- and MR cells. These results suggest that O6-methylguanine (O6-MeG) is involved in the induction of the biological effects of MNNG such as cytotoxicity, mutagenicity and SCEs, and also indicate that SCE induction does not always correlate with cell killing and mutation.     

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.     

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.     

Strand breaks arising from the repair of the 5-bromodeoxyuridine-substituted template and methyl methanesulphonate-induced lesions can explain the formation of sister chromatid exchanges

5-Bromodeoxyuridine (BrdU)-induced sister chromatid exchanges (SCEs) are mainly determined during replication on a BrdU-substituted template. The BrdU, once incorporated, is rapidly excised as uracil (U), and the gap is repaired with the incorporation of BrdU from the medium, which leads to further repair. During the second S period in BrdU medium, this process continues as the strand acts as template. Experiments suggest that 3-amino-benzamide (3AB) delays the ligation of the gaps formed after U excision, resulting in enhanced SCE levels during the second cycle of BrdU incorporation. When normal templates of G1 cells are treated before BrdU introduction with methyl methanesulphonate (MMS), 3AB in the first cycle doubles the MMS-induced SCEs but has no effect on them during the second cycle. When the BrdU-substituted template is treated with MMS in G1 of the second cycle, 3AB again doubles the SCEs due to MMS and also enhances the SCEs resulting from delays in ligation of the gaps following U excision in the BrdU-substituted template. The repair processes of MMS lesions that are sensitive to 3AB and lead to SCEs take place rapidly, while the repair process of late repairing lesions that lead to SCEs appear to be insensitive to 3AB. A model for SCE induction is proposed involving a single-strand break or gap as the initial requirement for SCE initiation at the replicating fork. Subsequent events represent natural stages in the repair process of a lesion, ensuring replication without loss of genetic information. G1 cells treated with methylnitrosourea (MNU) and grown immediately in BrdU medium rapidly lose the O⁶-methylguanine from their DNA and the rate of loss is BrdU-dose dependent. The rapid excision of the U lesions can explain the effect of BrdU concentration on SCE reduction following both MNU or MMS treatment.     

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.     

Effects of a tumor promoter and an anti-promoter on spontaneous and UV-induced 6-thioguanine-resistant mutations and sister-chromatid exchanges in V79 Chinese hamster cells

The effects of a tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) and/or an anti-promotor antipain (protease inhibitor) on spontaneous and ultraviolet-induced sister-chromatid exchanges (SCEs) and 6-thioguanine- resistant (6TG^r) recessive mutations were examined in V79 Chinese hamster cells in culture. TPA and/or antipain neither significantly altered base-line and UV-induced immediate SCE frequencies, nor decreased the level of delayed SCEs which persisted 6–7 days after irradiation. TPA and/or antipain appeared to enhance the recovery of UV-induced 6TG^r colonies at the plateau expression phase despite non-mutagenicity by themselves and unaltered metabolic co- operation. Thus, the results conceivably imply that the 6TG^r-recessive mutation expression, but not fixation, can be modulated at the cell level by the TPA and/or antipain. Our results, together with the recent results of Loveday and Latt, may argue against the notion that TPA enhances the antipain-suppressible SCEs as an index of mitotic recombination in relevance with a tumor-promotion mechanism.     

Gene mutation and sister chromatid exchange in Chinese hamster cells

The mutation in hypoxanthine phosphoribosyl transferase gene and the induction of sister chromatid exchange (SCE) were comparatively studied treating Chinese hamster ovary cells with the mutagens ethylmethanesulphonate. N-methyl-N'-nitro-N-nitrosoguanidine, Mitomycin C and X-ray. All the agents exerted strong mutagenic effects and showed a dose-dependent relationship for the induction of SCEs.     

Evidence That BRCA1- or BRCA2-Associated Cancers Are Not Inevitable

Inheriting a BRCA1 or BRCA2 gene mutation can cause a deficiency in repairing complex DNA damage. This step leads to genomic instability and probably contributes to an inherited predisposition to breast and ovarian cancer. Complex DNA damage has been viewed as an integral part of DNA replication before cell division. It causes temporary replication blocks, replication fork collapse, chromosome breaks and sister chromatid exchanges (SCEs). Chemical modification of DNA may also occur spontaneously as a byproduct of normal processes. Pathways containing BRCA1 and BRCA2 gene products are essential to repair spontaneous complex DNA damage or to carry out SCEs if repair is not possible. This scenario creates a theoretical limit that effectively means there are spontaneous BRCA1/2-associated cancers that cannot be prevented or delayed. However, much evidence for high rates of spontaneous DNA mutation is based on measuring SCEs by using bromodeoxyuridine (BrdU). Here we find that the routine use of BrdU has probably led to overestimating spontaneous DNA damage and SCEs because BrdU is itself a mutagen. Evidence based on spontaneous chromosome abnormalities and epidemiologic data indicates strong effects from exogenous mutagens and does not support the inevitability of cancer in all BRCA1/2 mutation carriers. We therefore remove a theoretical argument that has limited efforts to develop chemoprevention strategies to delay or prevent cancers in BRCA1/2 mutation carriers.     

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.     

Approximation of baseline and BrdU-induced SCE frequencies

In the present paper we have developed a new rationale and an experimental schedule to approximate the frequency of SCEs which occur independently of BrdU incorporation, namely, the baseline frequency of SCEs. The method used includes the analysis of SCE yields in second and third division chromosomes after BrdU-substitution for 1, 2, and/or 3 successive replication rounds in the presence of this thymidine analogue, leading to a set of ten different experimental results. As a result of formulating various mathematical equations and applying them to the data, an accurate estimation of the frequency of baseline (BrdU-independent) and BrdU-induced SCEs, can be made, thus avoiding the difficulties inherent in the current extrapolation methods. The conclusions are that 1) SCEs seem to be formed after DNA synthesis (by exchanging post-replicative DNA portions), but, obviously, very near to the replication fork and 2) that under our experimental conditions about 0.065 SCEs per picogram of DNA per cell cycle occur as a consequence of chromosome replication, this frequency being increased by BrdU-substitution. The methodology seems to be reliable enough to be used in other species and systems in order to compare baseline SCE frequencies.Abbreviations SCEs sister-chromatid exchanges - BrdU(BrdUrd) 5-bromodeoxyuridine - dTh(dThd) thymidine - ³H-dTh(³H-dThd) tritiated thymidine - FdU(FdUrd) 5-fluorodeoxyuridine - Urd uridine - FPG fluorescent plus Giemsa     

Synergistic action of cysteamine and BrdU-substituted DNA in the induction of sister chromatid exchanges

The effect of different BrdU-concentrations on the cysteamine-induced SCE-rate was investigated in V-79 Chinese hamster cell monolayer cultures. Both cysteamine and its auto-oxidation product cystamine act synergistically with BrdU in the induction of SCEs. A given concentration of these substances produces a low SCE-frequency at low BrdU-concentrations — but the incidence of SCEs is significantly increased at increased BrdU-concentrations. — Using one-cycle substitution experiments and the determination of the relative level of substitution by means of ³HBrdU-incorporation, this synergism was shown to depend on the BrdU incorporated in the DNA and on the extent to which this incorporation takes place.     

Auslösung der Blütenbildung durch Cycloheximid im Kurztag bei der Langtagpflanze Hyoscyamus niger

Summary Cycloheximide (CH) was applied selectively either to the shoot apex or by infiltration to the leaves of the long-day plant Hyoscyamus niger in order to investigate whether this inhibitor has an effect on the synthesis of a floral stimulus in the leaves. Treatment of the shoot apex with CH caused inhibition of the photoperiodic induction. In contrast, when CH was applied to leaves, initiation of flowering was observed under short-day conditions. The drug yielded optimum initiating effects at concentrations of 10^-5-3·10^-5 M, inducing flowering of almost 60% of the plants. Daily infiltration over a period of up to 4 days decreased the rate of flower initiation. The effect of CH was shown to be additive to a photoperiodic induction, even to a sub-threshold induction, but not to 2-thiouracil mediated induction. In no case did the presence of additional untreated leaves on the plants suppress CH-mediated flower induction. Treatment of the leaves with chloramphenicol (10^-6-2^-10^-4 M) or puromycin (5·10^-6-2·10^-4 M) caused no initiating response. The results are interpreted to mean that the presence of CH in the leaves may lead to the synthesis of a floral stimulus also under short-day conditions. This finding is similar to that reported previously in the case of the inductive effect of 2-thiouracil.

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Folgende Abkürzungen wurden verwendet 2-TU 2-Thiouracil - CH Cycloheximid - LT Langtag - DL Dauerlicht Herrn Prof. Dr. L. Brauner in Verehrung und Dankbarkeit zum 75. Geburtstag gewidmet.     

The effects of incorporated tritium and bromodeoxyuridine on the frequency of sister chromatid exchanges

Cells in third mitosis treated during the first cell cycle with ³H-TdR and during the next two cycles with BrdU (without ³H-TdR) show a typical pattern of chromosome differentiation which allows identification of sister chromatid exchanges occurring during the first (SCE₁, second (SCE₂) and third cycles (SCE₃). Chromosomes labeled only with ³H-TdR had the most SCEs; those labeled only with BrdU, the second highest number; and those labeled with ³H-TdR plus BrdU, the fewest. Since BrdU and ³H-TdR are well known inducers of SCEs, the relatively low frequency of exchanges produced by the combined action of these two compounds is paradoxical. — It is assumed that SCEs are generated by the abnormal recombination of double-strand DNA breaks occurring at the junctions between completely and partially duplicated replicon clusters. Thus, agents that induce absolute blocks to DNA fork displacement will favor the appearance of SCEs because double-strand breaks have more time to occur at junctions. Conversely, agents that inhibit the initiation of replication will decrease the probability of SCEs. Ionizing radiation delays the onset of cluster replication. Therefore, in ³H-TdR plus BrdU-substituted chromosomes the radiation from tritium may inhibit the appearance of BrdU-induced SCEs. Since the inhibition does not exist in chromosomes substituted only with BrdU, the frequency of SCEs in these elements is higher than in double-substituted chromosomes. During the first cell cycle the onset of cluster replication is normal. However, the incorporation of ³H-TdR in the replication fork may enhance the appearance of double-strand breaks, thus inducing a high frequency of SCEs.