SCE-inducing Lesions Research Articles

In vivo repair during G1 of DNA lesions eliciting sister chromatid exchanges induced by methylnitrosourea or ethylnitrosourea in BrdU substituted or unsubstituted DNA in murine salivary gland cells

The difference in efficiency of methylnitrosourea (MNU) and ethylnitrosourea (ENU) to induce SCE in early or late G1 was determined in synchronized murine salivary gland cells in vivo, as a measure of the capacity of this tissue to repair the lesions involved in SCE formation during G1. The repair during G1 was determined by treating the cells in early or late G1. Treatment was in the first cycle (G1 before incorporation of 5-bromodeoxyuridine (BrdU)) or in G1 of the second cycle (after a single round of BrdU incorporation). It was observed that 50% of the lesions induced by MNU that elicit SCE are repaired during G1. BrdU incorporation into DNA increases the sensitivity of the cell to SCE induction by MNU nearly 40%; however under this circumstance a slightly lower SCE frequency was observed in the cells exposed to MNU at early G1, indicating that during G1 only few lesions are repaired. The ENU-induced DNA-lesions involved in SCE production are nearly 100% persistent along G1; besides, a slight but significantly higher SCE frequency was observed in cells exposed at early G1, suggesting the formation of SCE-inducing lesions during G1. BrdU incorporation to DNA sensitizes the cell to SCE induction by ENU, increasing the SCE frequency to nearly to a 40%, although these additional lesions involved in SCE induction seem to be susceptible to repair during G1.

Sister chromatid exchange-inducing DNA lesions and depression of activation markers on the surface of cultured peripheral blood mononuclear cells after the addition of streptococcal pyrogenic exotoxins A and C.

Cultivation of peripheral blood mononuclear cells (PBMC) in the presence of streptococcal pyrogenic exotoxins (SPE) A and C resulted in a significant induction of sister chromatid exchange (SCE)-inducing DNA lesions. Concomitantly, the expression of interleukin-2 receptor alpha chain (IL-2R alpha chain), transferrin receptor (TfR), and major histocompatibility complex class II molecule HLA-DR on the surface of phytohemagglutinin-activated T cells from whole blood culture cells (WBCC) significantly decreased within 72 h, that is at least two cell cycles, whereas unstimulated T cells from WBCC did not express these markers but had lost their CD3 molecules, an effect reported to precede apoptosis as part of a T cell inactivation pathway. However, no apoptotic cells were observed within a cultivation period of 120 h. We observed clearcut differences in the responses towards SPE A in WBCC and isolated lymphocytes, since SPE A-treated lymphocytes showed an increase in the [3H]thymidine incorporation and did express IL-2R alpha chain and TfR on their cell surface. Regardless of the precise underlying mechanism, T cells from WBCC seem to be in a state of functional incompetence. The data presented here are the first to provide strong evidence that streptococcal toxins produce SCE-inducing DNA lesions in PBMC, an effect that might contribute to the process of immune cell lethality in streptococcal toxic shock-like syndrome and could be of pivotal importance in the pathogenesis of severe streptococcal disease.

Persistence during G1 of gamma ray- or mitomycin C-induced lesions eliciting SCE in murine salivary gland cells in vivo.

The efficiency of mitomycin C or gamma rays to induce SCE in early or late G1 was determined in synchronized murine salivary gland cells in vivo, as a measure of the capacity of this tissue to repair the lesions involved in SCE formation before S. The SCE frequencies induced by MMC in the first division (before BrdU incorporation) were significantly lower in the early G1 compared to the late G1, indicating some repair of SCE-inducing lesions. In the second division (after BrdU incorporation), there was no difference between SCE induced in early and late G1, indicating that MMC-induced lesions in such conditions are very persistent and not repairable during G1. The radio induced SCE frequency at early G1 was significantly lower than that observed in late G1, in cells irradiated after BrdU incorporation, suggesting that half of gamma ray-induced DNA lesions that elicit SCEs were repaired.

Sister-chromatid exchange (SCE) elimination kinetics in BrdUrd-treated cells irradiated with visible light

In the present paper we have characterized the ability of an irradiation with visible light of BrdUrd-containing chromosomes to induce sister-chromatid exchanges (SCEs). The experimental approach has include the use of post-treatments with oxygen, caffeine or benzamide during the pre-replicative period of the cell cycle in Allium cepa L. root-meristem cells exposed to visible light (VL) during early G1. Cells post-treated with benzamide immediately after VL showed higher SCE levels than those post-treated with either caffeine or oxygen. SCE-inducing DNA damage, repaired by a benzamide-sensitive step, appeared to be eliminated with a very fast kinetics. Thus, 15 min between the end of VL exposure and the beginning of benzamide treatment were enough for the cells to exhibit normal SCE levels. However, for the case of oxygen, that time was shown to be 2–3 h. Since these agents presumably act at different levels of the repair process, our results suggests that different SCE-inducing lesions were produced by VL exposure and/or that they were repaired at different times throughout the G1 phase. Finally, when cells irradiated at the beginning of the G1 phase were treated with either caffeine or benzamide during their G1/S traverse, SCE levels were significantly increased. These results were consistent with the occurrenceof a benzamide- and caffeine-sensitive survey mechanism, acting at the G1/S border, that would tend to reduce the SCE-inducing DNA damage before the S-phase.

Effects of liquid holding by l-ethionine on the SCE frequency induced by nitrogen mustard in human lymphocyte cultures

The highest SCE frequency induced by HN2 in PHA-stimulated human lymphocyte cultures is obtained when cells are treated immediately before the S phase, about 27–30 h after PHA stimulation. When HN2 treatment occurs during the G 1 phase, the induced SCE frequency is inversely related to the duration between treatment and the onset of the S phase, suggesting that removal of the SCE-inducing lesion occurs during G 1. High concentrations of l-ethionine (15–30 mM) inhibit cell-cycle progression in G 1. The SCE frequency induced by treatment with HN2 (0.5 μM) during liquid holding is always lower than the SCE frequency induced by treatment with HN2 after release of the cells from liquid holding. This result indicates that SCE-inducing lesions caused by HN2 are removed during the cell-cycle delay. Cultures treated with HN2 after release from 2–12 h of liquid holding in late G 1 show a higher SCE frequency than non-arrested, HN2-treated cultures. This effect is likely due to an enrichment of cells in S after release from liquid holding.

Inhibition of uracil-DNA glycosylase increases SCEs in BrdU-treated and visible light-irradiated cells

We have approached the study of the ability of different types of lesions produced by DNA-damaging agents to develop sister-chromatid exchanges (SCEs) by analyzing SCE levels observed in Allium cepa L cells with BrdU-substituted DNA and exposed to visible light (VL), an irradiation which produces uracil residues in DNA after debromination of bromouracil and enhances SCE levels but only above a certain dose. We have partially purified an uracil-DNA glycosylase activity from A. cepa L root meristem cells, which removes uracil from DNA, the first step in the excision repair of this lesion. This enzyme was inhibited in vitro by 6-amino-uracil and uracil but not by thymine. When cells exposed to VL, at a dose that did not produce per se an SCE increase, were immediately post-treated with these inhibitors of uracil-DNA glycosylase, a significant increase in SCE levels was obtained. Moreover, SCE levels in irradiated cells dropped to control level when a short holding time (less than 15 min) elapsed between exposure to VL and the beginning of post-treatment with the inhibitor. Thus, our results (1) showed that inhibitors of uracil-DNA glycosylase enhanced SCE levels in cells with unifilarly BrdU-substituted DNA exposed to visible light; (2) pointed to uracils and/or to some products of their repair as lesions responsible for SCE formation under our experimental conditions; and (3) indicated the existence of a very rapid repair of SCE-inducing lesions produced by visible light irradiation of cells with unifilarly BrdU-containing DNA.

Do the frequencies of sister chromatid exchanges in endoreduplicated mitoses provide a measure for lesion persistence and repair?

Endoreduplication was induced in V 79 cells using Colcemid. The concentration of Colcemid necessary to induce endoreduplication is about 1000 times higher than that needed to arrest mitoses or to induce ordinary tetraploid cells. Diplochromosomes with sister chromatid differentiation were obtained by adding BrdU for the duration of one cell cycle prior to the induction of endoreduplication. The induction of endoreduplication with Colcemid had no influence on the frequency of sister chromatid exchanges (SCEs). Treating the cultures with mitomycin C (MMC) before adding BrdU increased the percentage of endoreduplicated mitoses and also led to marked SCE induction. In the diplochromosomes, the frequencies of both twin SCEs (first cycle) as well as single SCEs (second cycle) were increased. It was also found that the SCE frequencies in mitoses after endoreduplication were lower than the values found in diploid and ordinary tetraploid metaphases of the same preparation. The possible conclusions concerning the lifetime of SCE-inducing lesions and the influence of repair processes are discussed.

Persistence of cyclophosphamide-induced damage in bone marrow as indicated by sister chromatid exchange analysis.

Various treatment protocols were used to investigate the time-dependent decrease in sister chromatid exchanges (SCEs) in bone marrow cells following treatment of C57Bl/6J X DBA/2J F1 (DB2F1) mice by i.p. injection of cyclophosphamide (15 mg/kg). The major factor in the time-related decrease in SCE-inducing lesions is the considerable cytotoxicity or selection of less highly damaged cells over successive cyclophosphamide post-treatment cycles. A constant rate of selection of 0.61 and 0.65%, respectively, was apparent over post-treatment cycles 1-2 and 2-3. In addition, second- and third-division SCE data produced by various protocols indicate persistence of a fraction of cyclophosphamide's SCE-inducing lesions for at least three post-treatment cycles. Comparison of the persistence of cyclophosphamide's SCE-inducing lesions with our previously reported data for diepoxybutane and ethyl carbamate reveals that the rate of repair of SCE-inducing lesions is inversely related to tumorigenic activities.

SCE induction is uncoupled from mutation induction in mammalian cells following exposure to ethylnitrosourea (ENU).

It has been suggested not only that sister chromatid exchange (SCE) induction might serve as a qualitative indicator of mutagenesis, but also that induced SCE frequencies are linearly related to induced mutation frequencies. The consistency of the relationship between SCEs and mutations was tested in the present work. Confluent Chinese hamster ovary (CHO) cells were exposed to ethylnitrosourea (ENU) and then held at confluency for various times prior to initiation of SCE and mutation assays. Cells held at confluency are typically thought to be arrested in their progression through the cell cycle, so that "S-dependent" processes such as fixation of mutations and formation of SCEs will not occur, while DNA repair processes might continue to operate. If repair processes reduce the number of SCE-inducing lesions during the holding period and, hence, reduce the subsequently determined SCE frequencies, then mutation frequencies should similarly be reduced if SCEs and mutations are related. It was observed, however, that induced SCE frequencies decreased exponentially with holding time, while mutation frequencies remained constant. Qualitatively similar results were obtained in log-phase cells. Cell cycle analysis demonstrates that confluent CHO cells can cycle, and ways are considered in which this might affect SCE and mutation frequencies. It is concluded that the decline in SCE frequency (with time) cannot be attributed solely to the presence of cycling cells in confluent cultures. It appears, therefore, that at least some forms of ENU-induced DNA damage that can lead to SCE were repaired and as such are distinct from those forms that are mutagenic. Thus SCEs are not necessarily related to mutations, because the two events may represent manifestations of different forms of DNA damage. Whether or not this represents a universal phenomenon that would hold true for agents other than ENU remains to be determined.

Removal and persistence of SCE-inducing damage in human lymphocytes in vitro.

The SCE frequency was studied in PHA-stimulated human lymphocytes exposed to various SCE-inducing agents in different stages of the cell cycle. Melphalan, HN2, MMS, and UV light were found to induce a higher SCE frequency in late G1 (18-24 hr after PHA stimulation) than in early G1 (1-6 hr after PHA) or G0 (before PHA stimulation). In contrast, CCNU induced more SCEs in early G1 than in late G1, and the adriamycin-induced SCE frequency was about the same after treatment in early and late G1. These results suggest that SCE-inducing lesions are being removed at different rates in human G1 lymphocytes. The removal of SCE-inducing HN2 lesions was found to be about 10 times more rapid in late G1 than in early G1, indicating the activation of a cross-link repair mechanism prior to DNA replication in human lymphocytes. Cells treated with MMS in the second G1 (after cultivation for about 55 hr in the presence of PHA and BrdUrd) showed a higher SCE frequency than cells treated with the same dose of MMS in the first G1. This result indicates that some type of interaction occurs between MMS damage and BrdUrd lesions in the DNA during replication, which leads to an enhanced induction of SCE. Analysis of SCEs induced during the 2 first vs. the third cell cycle in third-generation metaphases showed that most of the SCE-inducing damage caused by treatment with HN2 and melphalan in G1 of the first cell cycle are removed before the S phase of the third cell cycle, whereas damage caused by MMS and adriamycin seem to be more persistent. These observations suggest that the rate by which different types of SCE-inducing damage are removed or modified in resting (G0) of PHA-stimulated human lymphocytes can have a great influence on the SCE frequency. This is of practical importance in studies using SCE analysis to evaluate human exposure to suspected genotoxic agents in the environment.

O6-methylguanine, but not N7-methylguanine or N3-methyladenine, induces gene mutations, sister-chromatid exchanges and chromosomal aberrations in Chinese hamster cells

Induction of mutations to 6-thioguanine resistance, sister-chromatid exchanges (SCEs) and chromosomal aberrations were studied in V79 cells grown for 24 h in medium containing either O 6-MeG, N7-MeG, N3-MeA, guanine or adenine. N7-MeG and N3-MeA, at least in the concentration range tested, did not significantly increase the frequency of mutations, SCEs or aberrations. Exposure of cells to O 6-MeG within the concentration range of 1–12 μg/ml reduced cell survival and induced mutations and SCEs. The mutation frequency increased exponentially with dose, whereas the yield of SCEs increased linearly with dose. O 6-MeG also induced chromosomal aberrations, however relatively high concentrations and long periods between treatment and fixation were necessary to obtain a reasonable yield. With O 6-MeG concentrations up to 40 μg/ml, not more than 25% of the cells showed aberrations, but chromosomes were frequently uncoiled and showed a large number of gaps. Guanine and adenine induced neither mutations nor SCEs. Only adenine proved to be slightly clastogenic. The results indicate that O 6-MeG may be incorporated into DNA and give rise to promutagenic and SCE-inducing lesions. Differences of dose-response relationship, however, indicate that mutations and SCEs are not directly related and that different factors are involved in mutation induction and SCE formation after exposure to O 6-MeG. Furthermore, the results suggest that the probability that O 6-MeG will produce chromosomal aberrations is low as compared with the induction of mutations and SCEs.

Induction of sister-chromatid exchanges by chemotherapeutic drugs in spermatogonia of mice: effects of procarbazine, adriamycin, cyclophosphamide and mitomycin C

The chemotherapeutic drugs procarbazine (PCB), adriamycin (ADR), cyclophosphamide (CP) and mitomycin C (MMC) were evaluated in vivo for induction of sister-chromatid exchanges (SCEs) in differentiating spermatogonia of mice. There was a dose-dependent increase in SCE induced by the drugs. The lowest doses that enhanced the SCE rate to > twice baseline frequency were: 10 (PCB), 0.25 (ADR), 5 (CP) and 0.25 (MMC) mg/kg body weight. Based on the x-fold increase in SCE over baseline frequency, induced by the highest test dose (which permitted analysis of SCE), the drugs evaluated were ranked as follows: CP > MMC > PCB > ADR. The persistence of SCE-inducing lesions in spermatogonia was investigated by giving a treatment of CP (20 mg/kg body weight), 6 and 9 days before completion of 5-bromodeoxyuridine (BrdUrd) administration. Within 6–9 days, the SCE rate returned to the baseline level.