Influence of incorporated 5-bromodeoxyuridine on the frequencies of spontaneous and induced sister-chromatid exchanges, detected by immunological methods

We have utilized monoclonal antibody against BrdUrd to detect sister-chromatid exchanges in CHO cells. This technique allows detection of SCEs at very low levels of BrdUrd incorporation. At incorporation level of 0.5%, a frequency of about 2 SCEs/cell/cycle was found. In a UV-sensitive mutant (43-3B) which has an increased spontaneous frequency of SCEs, it is found that this increase is due to incorporated BrdUrd. In MMS- and MMC-treated cells, an influence of BrdUrd on the frequencies of induced SCEs was found only when high concentrations of mutagens were employed.     

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.     

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.     

5-bromodeoxyuridine tablets with improved depot effect for analysis in vivo of sister-chromatid exchanges in bone-marrow and spermatogonial cells

An improved 5-bromodeoxyuridine (BrdU) tablet technique for observation in vivo of SCE in mouse bone-marrow and spermatogonial cells is described.BrdU tablets were coated with agar as protecting barrier before subcutaneous implantation into mice. In comparison with the original tablets, the agar-coated tablets provided a slower and more uniform delivery of BrdU to the animals. This was corroborated (1) by recovering the undissolved portion of tablets at 1–2-h intervals, and (2) by quantitative determination of the BrdU levels in blood with the help of an analytical HPLC technique.The time required for complete dissolution of the coated tablets was considerably longer than that for the original tablets. This means that the dose of BrdU required for observation of SCE in mouse bone-marrow cells can be reduced accordingly. By using these modified tablets, therefore, undesired effects of high doses of BrdU on mutation (base-line SCE frequency) as well as on cellular replication and proliferation can be diminished.Moreover, the improved depot effect of the modified tablets facilitates the differential labeling of sister chromatids in mouse spermatogonia, a tissue containing cells with a relatively long DNA synthesis period.     

Fate of DNA lesions that elicit sister-chromatid exchanges

Using 3-way differential staining (TWD) of sister chromatids, the fate of DNA lesions involved in sister-chromatid exchange (SCE) formation was determined in murine bone marrow cells in vivo, after treatment with either mitomycin C (MMC) or cyclophosphamide (CP). Both MMC (2.6 mg/kg b.w.) and CP (7 mg/kg b.w.) induced an SCE frequency near the expected in the 2 subsequent cell divisions, but the frequency of SCE occurring at the same locus in successive cell divisions was substantially lower than expected. The results are compared with previous data obtained after exposure to gamma-rays. A model of SCE induction is proposed.     

Incorporation of 5-bromodeoxyuridine into DNA in newborn rat tissues

The incorporation of bromodeoxyuridine (BUdR) into newborn rat tissue DNA has been determined after i.p. injection of 5-bromo-2′-deoxy[6-³H]uridine. Incorporation of the unchanged nucleoside was shown by hydrolysis and ion exchange chromatography of extracted DNA. In all tissues examined, more than 90% of the radioactivity incorporated was in the form of bromodeoxyuridine.     

Uncoupling of the induction of mutations and sister-chromatid exchanges by the replication of 5-bromouracil-substituted DNA

The REP mutagenesis protocol, which involves the replication of 5-bromouracil (BrUra)-substituted DNA in the presence of deoxyribonucleoside triphosphate (dNTP) pool imbalance, has been shown to induce both mutations and sister-chromatid exchanges (SCEs) in Chinese hamster ovary (CHO) cells. However, when a Syrian hamster melanoma-derived cell line, called 2E, which was selected for its ability to replace all of the thymine residues in DNA with BrUra, was subjected to the REP mutagenesis protocol, the correlation between the induction of mutations and SCEs was no longer observed. The 2E cells were found to be much more sensitive to the induction of mutations by REP mutagenesis than were the CHO cells. This increased sensitivity to REP mutagenesis was found to correlate with increased perturbations of the dNTP pools that have been shown to be involved in the mutagenic mechanism of this protocol. In contrast, when the induction of SCEs by the REP protocol was measured, it was found that although a baseline level of SCEs was detected in 2E cells, no significant induction of SCEs due to dNTP pool perturbation was observed. It was shown that high levels of SCEs were readily induced in 2E cells by other agents, e.g. mitomycin C. A model, which discusses the fate of mismatched bases thought to be generated by the REP mutagenesis protocol as the determining factor for the induction of mutations of SCEs, is proposed to explain the uncoupling of mutagenesis and SCE induction in 2E cells.     

On the nature of sister-chromatid exchanges in 5-bromodeoxyuridine-substituted chromosomes

Sister-chromatid exchanges (SCE) were studied in allium cepa L. meristematic cells at the second and third divisions after BrdUrd-substitution during just the first or during the second and third cycles, respectively. The observed SCE nonreciprocal/reciprocal ratios detected at the third division in both experiments, as well as comparison of the lowest SCE frequency observed per cycle and expressed per picogram of DNA with data from different species expressed accordingly, strongly suggest that most of the exchanges detected in BrdUrd-substituted chromosomes are BrdUrd-dependent events. Hypotheses suggesting some different mechanisms are discussed to explain the formation of these BrdUrd-dependent SCEs.     

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.     

Frequency and distribution of sister-chromatid exchanges in a case of Fanconi's anemia.

In lymphocytes of a 7-year-old boy with Fanconi's anemia the frequencies and sites of sister-chromatid exchanges (SCE) were studied with the BrdU-Giemsa method. The average frequency of SCE (8.8 per metaphase) and the inter- and intrachromosomal distribution of SCE was not significantly different from the controls.     

Actions of amino-beta-carbolines on induction of sister-chromatid exchanges

Synthetic 3-aminoharman and 3-aminonorharman (amino-beta-carbolines) caused slight but definite induction of sister-chromatid exchanges (SCEs) in human lymphoblastoid cells NL3 and Chinese hamster cells CHO-K1. These amino-beta-carbolines are ranked between 2-amino-alpha-carboline and 2-amino-6-methyl-9a-aza-delta-carboline (Glu-P-2) and much lower than 3-amino-gamma-carbolines (Trp-P-1 and 2) in inductive activity. 1-Amino-beta-carboline, harman and norharman had very weak, if any, SCE-inducer activity. Norharman had a synergistic effect with aromatic amines such as Trp-P-2 and aniline on SCE induction, while 3-aminoharman suppressed SCE induction by more potent inducers such as Trp-P-2 and benzo[a]pyrene.     

Effects of amino acids on sister-chromatid exchanges.

The effects of 10 amino acids on sister-chromatid exchange (SCE) frequency in human peripheral blood lymphocytes (PBL) and six amino acids on the SCE frequency in root tip cells of Hordeum vulgare were studied. Alanine (Ala), glycine (Gly), phenylalanine (Phe), valine (Val), histidine (His) and serine (Ser) induced a significant increase in SCE in PBL but threonine (Thr), isoleucine (Ile), lysine (Lys) and arginine (Arg) did not. Ala, Gly, Thr, Ile and Val induced a significant increase in SCE in root tip cells of Hordeum vulgare but Lys did not. The effect of Lys and bromodeoxyuridine (BrdU) on SCE levels in PBL and the interaction between them were also studied. The results show that Lys can inhibit the SCE induced by BrdU.     

Effects on chromosomal proteins in sister chromatid differentiation by incorporation of 5-bromodeoxyuridine into DNA

When fixed metaphase chromosomes of human lymphocytes grown in the presence of BrdUr for two cell cycles were stained with amino group-specific 2-methoxy-2,4-diphenyl-3(2H)furanone (MDPF) after a previous extraction of DNA, sister chromatids showed a light-independent differential staining. Although more faintly differential, a similar staining pattern being just the reverse of the DNA-specific DAPI pattern was obtained without prior removal of DNA. We conclude that the chromatid containing bifilarly BrdUr-substituted DNA has a higher protein content, at least after fixation, than the chromatid containing unifilarly BrdUr-substituted DNA. Possibly, a higher degree of BrdUr substitution leads to a tighter binding of chromosomal proteins. In line with this suggestion we found a markable difference in DNA extractability of BrdUr-containing and normal cytological preparations.     

The incorporation of 5-bromodeoxyuridine into DNA of the area opaca vasculosa of chick embryos

The incorporation of 5-bromodeoxyuridine (5-BrdUrd) into DNA of the area opaca vasculosa (AOV) of chick embryos during organ culture was measured. The AOV from blastoderms of the definitive primitive streak stage will not form red cells in the presence of BrdUrd while the AOV of 1–3 somite blastoderms is unaffected by the presence of 5-BrdUrd. About 90% of the original non-density labeled DNA can replicate in the presence of 5-BrdUrd if the tissues come from the younger sensitive embryos, but only 65% of the original DNA will replicate from tissues of older insensitive embryos. Tissues from embryos of both ages replace about 80% of the thymidine by BrdUrd in each newly synthesized strand of DNA; tissues from embryos of both ages will form DNA of hybrid density after one cell generation, and will also form double-heavy DNA after longer periods of culture in the presence of 5-BrdUrd. During recovery from 5-BrdUrd inhibition during a thymidine chase, the density-labeled DNA is replicated so that the new DNA of normal density is formed, but the original heavy 5-BrdUrd containing strands are conserved. It is suggested that inhibition of red cell formation by 5-BrdUrd may occur by incorporation of 5-BrdUrd into DNA of endoderm cells, rather than by acting only directly on red cell precursors.     

Differences between diploid and transformed human cells in the incorporation of 5-bromodeoxyuridine into DNA

In diploid human cells, the DNA precursor pool equilibration times for exogenous thymidine are about twice those for the thymidine analogue 5-bromodeoxyuridine (BUdR); in cells that were either transformed chemically or derived from malignant tumours, the pool equilibration times are the same for thymidine and 5-bromodeoxyuridine and are closer in value to the shorter (bromodeoxyuridine) times of the diploid cells. Thymidine, if present in the culture medium with BUdR, is incorporated into DNA preferentially in diploid cells (by 2 or 3 to 1). Discrimination against bromodeoxyuridine is evident within 2 h of incubation of the two precursors with diploid cells, but is not observed even after 24 h in any of the transformed cell lines tested. Experiments were performed to test the effect of inhibitors of the mammalian DNA polymerases alpha (N-ethylmaleimide) and beta (incubation of cells at 45 °C) upon the ability of cells to synthesise DNA and to incorporate thymidine preferentially when present with equimolar BUdR. In diploid cells, overall in vivo DNA synthesis is more sensitive to N-ethylmaleimide and more resistant to 45 °C treatment than is DNA synthesis in the transformed cell lines. N-Ethylmaleimide decreases the capacity of diploid cells to discriminate against BUdR, whereas heating increases it. Transformed cells treated with N-ethylmaleimide remain unable to discriminate against BUdR; some transformed lines, when heated at 45 °C, become less incapable of such discrimination.     

Possible effect of the cell synchronization method on the pattern of 5-bromodeoxyuridine incorporation into early synthesized DNA

Synchronously and asynchronously growing chick embryo fibroblasts have been used to study the pattern of 5-bromodeoxyuridine (BrdU) incorporation into DNA. In the synchronous cell system, the density of unifilarly substituted DNA is about 0.010 g/ml higher during first half of S phase than during second half of S phase. The density of unifilarly substituted DNA isolated from asynchronously growing cells is similar to that of DNA synthesized during the second half of S phase of synchronously growing cells for a given concentration of analogue in culture medium. Reassociation kinetics experiments have shown the oversubstitution to occur at the level of early synthesized repeated and/or intermediate DNA sequences. It is then assumed that the oversubstitution is due to some metabolic changes caused by the synchronization procedure itself. As BrdU incorporation into early replicating DNA is known to induce alterations of the cell metabolism, the implication of this phenomenon is discussed at the level of the inhibition of transformation which takes place when chick embryo fibroblasts are infected with Rous sarcoma virus during G1 and subsequently treated with BrdU during early S phase.