Sister chromatid exchange induction and persistence in peripheral blood and spleen lymphocytes of mice treated with ethylnitrosourea

The induction and persistence of sister chromatid exchanges (SCEs) were studied in peripheral blood and spleen lymphocytes of mice given a single i.p. injection of ethylnitrosourea (ENU) of 100, 350, or 600 muMoles ENU/kg. SCE frequencies were measured on days 1, 3, 5, and 7, and at seven additional times up to 172 days post-injection. SCEs were analyzed statistically by comparing the mean frequencies as well as the distribution of SCEs per cell at each time. The latter approach was based on a non-parametric method of identifying high frequency cells (HFCs). The SCE frequencies and proportion of HFCs in each dose and tissue remained elevated for up to 172 days following treatment, although the degree and statistical significance of the increase varied according to the tissue, dose, and statistical test employed. The SCE frequencies were found to oscillate during the first week. Following this, however, the return of the SCE frequencies to control levels was fit to a linear regression model with time as the only independent variable. The persistence of SCE-forming lesions was found to be dose-dependent for the spleen but not for blood. Within each dose the persistence of SCE-forming lesions was significantly greater for the blood relative to the spleen. The results emphasize that tissue, dose, and time since exposure are important factors to consider when quantifying SCEs in vivo; analysis of high frequency cells may be a more sensitive method of detecting exposure than the t-test; and a single determination of SCE frequencies may not be sufficient to quantitatively assess genotoxic damage in the first week following exposure.     

Mechanism of in vivo sister-chromatid exchange induction by 5-azacytidine

The aim of the present study was to explore the in vivo mechanism of sister-chromatid exchange (SCE) induction by 5-azacytidine (5-azaC) in murine bone marrow cells. Experiments were performed to examine SCE induction in response to different doses of 5-azaC as well as several exposures. Additionally, we examined the persistence of SCE induction and the effect of bromodeoxiuridine (BrdU) incorporation. Sister-chromatid differentiation was obtained by injecting mice intraperitoneally with BrdU absorbed to activated charcoal. Before BrdU injection, different doses of 5-azaC were administered intraperitoneally either singly or in multiples. Colchicine in an aqueous solution was administered subcutaneously 22 h after BrdU injection. Two hours later, animals were sacrificed by cervical dislocation and both femurs were dissected. Bone marrow cells were processed to obtain chromosome preparations, which were stained by the fluorescence plus Giemsa method. Results indicate that 5-azaC caused SCE, albeit to a limited extent. In order to discern whether the limitation was due to cytotoxicity or to partial 5-azaC incorporation, we administered multiple sub-toxic doses of 5-azaC. This treatment increased 5-azaC incorporation and reduced cytotoxicity, but did not raise SCE frequency, indicating that the limitation was not due to either of the two factors mentioned above. SCE frequency induced by 5-azaC persisted for at least eight cell divisions, confirming that this agent had caused inhibition of DNA methyltransferase and subsequently the reduction of DNA re-methylation, which in turn induced the expression of a number of SCE-prone sites. Finally, SCE induction in response to 5-azaC was completely dependent on BrdU incorporation. The data allow us to conclude that 5-azaC causes SCE to a limited extent; limited SCE induction was not due to the direct effect of incorporation or cytotoxicity of 5-azaC, but rather the generation of a number of SCE-prone sites, the expression of which persists for at least eight cell divisions and is dependent on BrdU incorporation.     

Repairability during G1 of lesions eliciting sister chromatid exchanges induced by methylmethanesulfonate or ethylmethanesulfonate in bromodeoxyuridine-substituted and unsubstituted DNA strands

The repairability during G(1) of DNA lesions eliciting sister chromatid exchanges (SCE) induced by methylmethanesulfonate (MMS) and ethylmethanesulfonate (EMS) in BrdU-substituted and unsubstituted DNA strands was determined in murine salivary gland cells in vivo. The SCE frequency was determined after exposure to MMS or EMS during early and late G(1) in the first or second cell division. The inducibility and repairability of SCE-eliciting lesions during G(1) in BrdU-substituted and unsubstituted strands were estimated considering that in the first division induction occurs on the unsubstituted strand and during the second division in one unsubstituted and one BrdU- substituted DNA strand. The results indicate that DNA lesions induced by MMS are 50% repaired in both the BrdU-substituted and unsubstituted strands and those induced by EMS are 60% repaired in the unsubstituted strand but only 20% in the BrdU-substituted strand. The increase in sensitivity of the BrdU-substituted strand to SCE induction with respect to the unsubstituted strand was 155 and 45% for MMS and EMS, respectively. These results imply that SCE-inducing lesions produced by MMS and EMS are only partially repaired and that BrdU incorporation could sensitize DNA not only to the induction of lesions eliciting SCE, but also to the induction of non-repairable lesions.     

The in vivo induction of sister chromatid exchange by the demethylating agent 5-aza-2'-deoxycytidine

Previously, we observed that the demethylating agent 5-azacytidine (azaC) induces a constant and limited low frequency of sister chromatid exchange (SCE), seemingly due to a limited number of SCE-prone sites whose expression is related to DNA demethylation. An alternative explanation for the low frequency of SCE induction may be its inefficient incorporation into DNA, as it has a higher incorporation into RNA. The aim of the present work is to determine if the frequency of SCE induction is increased after exposure to 5-aza-2'-deoxycytidine (azadC), a compound with the same mechanism of demethylation as azaC but more efficiently incorporated into DNA. Groups of mice were treated with 2.2, 4.4, 6.6 and 8.8 μmol azadC per kilogram body weight, and the SCE frequency, the mitotic index and the average generation time were determined after two cell division cycles. The dose-response data of SCE induction showed two components: (i) a dose-dependent increase between 0 and 4.4 μmol and (ii) almost a same level of two SCEs per cell at 4.4 and 8.8 μmol. Although azadC is incorporated more efficiently into DNA, as shown by a lower dose required for a maximal effect, the highest frequency of SCE induction is similar to that observed with azaC. These results indicate that the low incorporation of azaC into DNA seems not to be the factor that limits SCE induction, but the limited number of specific SCE-prone sites in demethylated DNA. Perhaps, there are a restricted number of sites prone to homologous recombination due to DNA demethylation.     

Effects of acute and chronic administration of mitomycin C on the induction of sister chromatid exchanges in vivo

Frequencies of sister chromatid exchanges (SCE) were analyzed in bone marrow cells of mice injected with mitomycin C (MMC) both before and during infusion with bromodeoxyuridine (BrdU). Administration of MMC at 1, 6.5, and 13 hours after the onset of BrdU infusion resulted in the induction of approximately 45 SCE/cell, independent of time of administration. When MMC was injected 26 hours prior to BrdU infusion, only baseline levels of SCE were noted. The effects of multiple doses of MMC (chronic administration) were examined in mice treated with 1–5 mg/kg on a weekly or bimonthly basis. SCE analysis was performed one week after the final injection. At all doses and with all treatment regimes, SCE frequencies did not differ from control levels. The results indicate that most or all MMC-induced DNA damage that results in SCE formation is removed in a single cell cycle after its administration.     

Growth rate and sister chromatid exchange (SCE) incidence of bone marrow cells in Acute Myeloblastic Leukemia (AML)

The sister chromatid exchange (SCE) incidence and growth kinetics have been studied by means of an in vitro bromodeoxyuridine (BrdU) chromosome labeling method in the bone marrow cells of 17 acute myeloblastic leukemia (AML) patients with only diploid cells at diagnosis, remission, and relapse of the disease. At diagnosis, the cells tended to exhibit a low SCE frequency as compared to that during remission. An increased SCE frequency was observed after chemotherapy during remission or relapse. At diagnosis and relapse, when leukemic blast cells predominated in the marrow, they were characterized by the predominance of cells that had undergone only one cell cycle after BrdU exposure. In contrast, the marrow cells during remission tended to resemble the control pattern of growth kinetics, with a predominance of cells undergoing second and third cell cycles in the presence of BrdU. These results suggest that the growth rate of leukemic and nonleukemic cells is different, and that chemotherapy can cause an increased SCE frequency in the marrow cells of AML patients irrespective of the state of the disease.     

Occurrence in vivo of sister chromatid exchanges at the same locus in successive cell divisions caused by nonrepairable lesions induced by gamma rays

The capacity of lesions induced by gamma radiation to produce sister chromatid exchanges (SCE) in successive divisions in mouse bone marrow cells in vivo was evaluated using a protocol for the three-way differentiation of sister chromatids. Evidence was obtained that exposure to gamma radiation induces DNA lesions that result in the formation of SCE at the same locus in two successive cell divisions. The relevance of this observation with respect to DNA repair and mutagenesis is discussed.     

Sister chromatid exchange induced by short-lived monoadducts produced by the bifunctional agents mitomycin C and 8-methoxypsoralen

To see if DNA crosslinks are involved in the induction of sister chromatid exchange (SCE), Chinese hamster ovary cells were exposed to two bifunctional alkylating agents, mitomycin C and 8-methoxypsoralen, and their monofunctional derivatives, decarbamoyl mitomycin C and angelicin. The data indicate that monoadducts, rather than crosslinks, are responsible for SCE formation. Furthermore, all agents but angelicin produced short-lived lesions that led to SCEs in the first period of DNA replication after treatment (twin SCEs), but not in the second (single SCEs). In contrast, angelicin, like methyl methanesulfonate and N-acetoxyacetylaminofluorene, produced lesions that lasted more than one cycle, indicating that several different types of DNA lesions are capable of SCE induction.     

Proliferative characteristics and sister chromatid exchange (SCE) of colony-forming cells (CFU-S) in acute myelogenous leukemia

Sister chromatid exchange (SCE) and cell cycle progression of colony-forming cells (CFU-S) from patients with acute myelogenous leukemia were studied using bromodeoxyuridine (BrdU) incorporation and sister chromatid differential staining. The mean SCE rate of CFU-S was 7.99, which is similar to that reported for human peripheral blood lymphocytes. Cultures treated with BrdU at culture initiation and then harvested 72-120 hr later indicated a cell cycle time (TC) of approximately 60 hr. However, cells allowed to proliferate for 72-120 hr prior to the addition of BrdU showed TC values of 12-36 hr. Analysis of serial cultures from one patient revealed a heterogeneous population with a mean TC of 19.5 hr. These cell cycle times are considerably shorter than previously published reports and suggest that leukemic cells have the potential to divide rapidly.     

The longevity of chemically induced sister chromatid exchanges in chinese hamster ovary cells

The persistence of the lesions leading to sister chromatid exchanges (SCEs) following acute exposure of Chinese hamster ovary (CHO) cells to direct-acting chemical mutagens was measured. The results revealed that these lesions (and consequently SCEs) are rapidly eliminated from cells. SCE levels fell to near control values by the third or fourth day (six and eight cell cycles, respectively) following exposure of CHO cells to quinacrine mustard (QM) and mitomycin C (MMC). In contrast, cells exposed to methyl methanesulfonate (MMS) showed a small but significant increase in SCE level over control up to and including the fifth day following exposure (approximately ten cell cycles), suggesting that the behavior of these lesions in cells is influenced, at least in part, by the mechanism by which a specific agent interacts with DNA. The possibility that the decline in SCE level was due to the loss of cell populations with high numbers of exchanges was eliminated by the assessment of cloning efficiencies of treated and control cultures.     

Molecular dosimetry of sister chromatid exchange induction in 9L cells treated with 6-thioguanine

The induction of sister chromatid exchanges (SCE) in 9L cells treated with 6-thioguanine (6-TG) has been investigated. A 24 h treatment with 0.2 microM 6-TG induced approximately 28 SCE/metaphase. The dose-response curve was linear at doses below 0.2 microM and had a slope of 139 SCE/metaphase/microM 6-TG. At concentrations of 0.023 to 1 microM, incorporation of 6-TG in DNA was linear with dose. The slope of the dose-response curve was 4135 mumol 6-TG/mol DNA/microM 6-TG. Comparison of these results with those obtained in our previous studies of the monofunctional alkylating agent ethylnitrosourea and the bifunctional alkylating agent 3-(4-amino-2-methyl-5-pyrimidinyl)methyl-1-(2-chloroethyl)-1-nitrosourea suggest that to accurately estimate the effectiveness of particular DNA modifications at inducing SCE, the extent of formation of these DNA modifications must be known.     

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.     

Different effectiveness of 4-nitroquinoline-1-oxide, mitomycin C and ethyl methanesulfonate to induce lesions in DNA leading to sister chromatid exchange throughout successive cell cycles in Chinese hamster ovary cells.

The present study was carried out in Chinese hamster ovary cells in order to determine whether lesions induced by three different mutagens, namely 4-nitroquinoline-1-oxide (4-NQO), Mitomycin C (MMC) and Ethyl methanesulfonate (EMS), can persist for more than one cell generation leading to sister chromatid exchanges (SCE) or, alternatively, they are efficiently repaired during the next replicative period after treatment. In order to accurately score the number of SCEs arising during the first (S1), second (S2) and third (S3) DNA synthetic periods, third-cycle (M3) metaphases showing three-way differential (TWD) staining were analyzed. Our results show that, even though the three compounds tested were efficient in increasing the yield of SCE, the frequency of SCE was more dramatically increased after MMC treatment. Differences were also observed among the three mutagens with regard to the persistence of the lesions leading to SCE throughout successive cell generations. EMS-induced lesions appeared as more persistent than those induced by MMC. However, most of the damage induced by the UV mimetic agent 4-NQO seems to be efficiently repaired after the first round of DNA replication following treatment with the drug.     

Aging and sister chromatid exchange. VIII. Effect of the aging environment on sister chromatid exchange induction and cell cycle kinetics in Ehrlich ascites tumor cells. A brief note

To examine the effect of an aging environment on sister chromatid exchange (SCE) induction, Ehrlich ascites tumor (EAT) cells were introduced into young and old C57BL/6J mice. Background SCE levels were not significantly different in either EAT cell or normal bone marrow cell populations between young and old animals. Despite a decline in SCE induction in bone marrow cells in older mice at high mitomycin C concentrations, SCE induction in EAT cells was not significantly affected by the age of the animal. These findings suggest that the aging environment may not play a major role in the diminished SCE induction observed in old cell populations.     

The effect of aging on sister chromatid exchange.

The advent of the bromodeoxyuridine(BrdU)-differential staining techniques has greatly facilitated the detection of sister chromatid exchanges (SCE). These SCE have been demonstrated to be an accurate reflection of DNA damage both in vitro in cultured cells and in vivo in mouse and rate bone marrow and spleen cells. In this review, we examine the effect of cellular aging on both baseline and mutagen-induced SCE levels. In all systems examined, aging did not appear to significantly affect the baseline levels of SCE. However, in human fibroblast cultures we have found a significant decrease in the levels of mutagen-induced SCE as a function of both in vitro passage level (in vitro aging) and the age of the cell culture donor (in vivo aging). In addition we have found a similar decrease in mutagen-induced SCE levels in both mouse and rat bone marrow cells and mouse spleen cells where examinations were performed entirely in vivo. Diminished mutagen-induced SCE levels were obtained with a wide variety of agents including mitomycin-C, cyclophosphamide, adriamycin, ethyl methanesulfonate and N-acetyl-2-acetoxyamino-fluorene. These decreased SCE levels were accompanied by increased frequencies of chromosomal aberrations in the older cell populations. If SCE represents a form of DNA repair as has been suggested by several investigators, our finding would indicate impaired DNA repair occurring in old cells.     

Sister chromatid exchange induction near the baseline with low doses of the alkylating agent CCNU

Sister chromatid exchange (SCE) and cell-cycle kinetics were examined at near-baseline levels in human peripheral lymphocytes exposed to low doses of the potent SCE inducer 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), in vitro. A preliminary SCE dose-response curve was determined with a broad range of doses of CCNU using a single donor. SCE induction was approximately linear over the entire dose range from 5 to 200 microM CCNU. Cell cycle kinetics were retarded in a dose-dependent manner. A second dose-response curve from the same donor was constructed using several doses of CCNU between 0.5 and 10 microM to evaluate linearity and uniformity of SCE response near baseline levels. SCE induction was approximately linear between 1.0 and 10.0 microM CCNU. Finally, SCE and cell-cycle kinetics were examined in 12 donors at doses of 1.0, 5.0, and 10.0 microM CCNU to evaluate the reproducibility of near-baseline SCE induction over a range of subjects. Cell-cycle kinetics were retarded at all three doses with a highly significant increase in SCE frequencies at 5.0 and 10.0 microM CCNU. These data suggest that increases in SCE less than twice background can be reliable indicators of genotoxic exposure.     

X-Irradiation of G1 CHO Cells Induces SCE Which are Both True and False in BrdU-Substituted Cells But Only False in Biotin-dUTP-Substituted Cells

The SCE-test is widely used in genetic toxicology and therefore knowledge of the contribution of BrdU to the formation of spontaneous and induced SCE is of great importance. The present study was undertaken to analyse the role of BrdU in X-ray-induced SCE. If SCE resulted from inversions, rings and double minutes (RDM) would be the asymmetrical counterparts of SCE and should therefore have the same frequencies. Dose–effect relationships of SCE and RDM show that the frequencies of SCE are much higher than those of RDM. We conclude that only a few SCE may represent inversions. In a second set of experiments, endoreduplications were induced in cells irradiated either before or after labelling with BrdU. Analysis of SCE in endoreduplicated chromosomes allows the discrimination of the cell cycle in which they originated. The results show that SCE are only induced in the first cell cycle following irradiation of BrdU-substituted cells, indicating that labelling with BrdU is a necessary prerequisite for the formation of SCE. In order to test this directly, radiation-induced SCE frequencies were studied in cells prelabelled with BrdU or biotin-dUTP in a third set of experiments. The structure of biotin-dUTP suggests that, in contrast to BrdU, it does not give rise to radicals during irradiation. Significantly lower frequencies of SCE were observed in biotin-dUTP-substituted cells than in BrdU-labelled cells. Calculations show that nearly all SCE induced in biotin-dUTP-labelled chromosomes can be explained by chromosomal aberrations (false SCE). In contrast to this, most SCE induced by X-rays in BrdU-labelled cells are not due to chromosomal aberrations, but result from S-dependent lesions (true SCE). This clearly points towards radiation damage in BrdU-moieties as the source of DNA lesions leading to SCE.     

Different effects of mutagens on sister chromatid exchange induction in three Chinese hamster cell lines

The sister chromatid exchange (SCE) induction of mutagens with different mechanisms of action was comparatively investigated on permanent cell lines of the Chinese hamster (CHO, V-79, and DON) with and without exogenous metabolic activation and with the use of various experimental protocols. CHO and V-79 cells were treated with ethylmethanesulfonate (EMS), a direct mutagen; with the two indirect mutagens cyclophosphamide (CP) and benzo[a]pyrene (BP); as well as with the radical former hydrogen peroxide (H2O2) and hydroxyurea (HU), an inhibitor of DNA synthesis. Aside from an increased basal SCE level and a higher bromodeoxyuridine (BrdUrd) sensitivity, there was no decisive difference between CHO and V-79 cells. However, there was a distinct relationship between SCE induction and the experimental protocol used, which was most pronounced after HU treatment. Neither cell line was able to metabolize the indirect mutagen BP. Only in CHO cells did CP lead to increased SCE frequencies. However, in all cases, the simultaneous application of S9 mix produced a distinct SCE induction. In contrast, BP caused SCE induction in DON cells, whereas CP was not metabolized. The reason for these findings must obviously be sought in the metabolization of CP and BP via different monooxygenase systems, whose activity can differ in these permanent cell lines. One notable finding was that the number of SCE induced by H2O2 could be distinctly reduced by the simultaneous application of S9 mix. This effect can be explained by the fact that S9 mix contains H2O2-degrading enzymes. The results indicate that closely related cell lines differ in their capability for inducing SCE and that investigations of SCE inductions performed on only one cell line do not necessarily produce a representative response.     

The restriction endonuclease AluI induces sister chromatid exchange in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells were exposed to the restriction endonuclease AluI in the presence of 1.1 M glycerol. Chromosomal aberrations and sister chromatid exchange (SCE) were scored in first post-treatment metaphases following recovery times of 6-18 h. At all recovery times chromosomal aberrations were induced by the enzyme. In AluI-treated damaged cells significant elevations of SCE frequencies were found after recovery times of 12-18 h. These results indicate that SCE, unlike chromosomal aberrations, are induced only in late G1 and early S phase of the cell cycle. The lesions producing SCE are postulated as DNA single-strand breaks and gaps induced by AluI in canonical structures of DNA and in DNA-RNA hybrids.     

Low frequency noise and whole-body vibration cause increased levels of sister chromatid exchange in splenocytes of exposed mice

Chronic exposure to low frequency (LF) noise and whole-body vibration (WBV) induces both physiological and psychological alterations in man. Recently, we have shown that long-term occupational exposure to LF noise and WBV produces genotoxic effects in man expressed as an increase in sister chromatid exchange (SCE) levels in lymphocytes. The objectives of the present study were to investigate whether the observed effect could be reproduced in a murine model and, if so, which of the agents, LF noise alone or in combination with WBV, would be instrumental in the SCE induction. SCEs were analyzed in spleen lymphocytes of mice exposed to LF noise alone and in combination with WBV for 300 and 600 hr. An effect at the cell cycle kinetics level was also investigated. The results revealed significant increases in the mean SCE number per cell and in the proportion of cells with high frequency of SCEs (HFCs) in lymphocytes of mice submitted to combined noise and WBV over controls. No significant differences were found between single noise-exposed and control mice. A cell cycle delay was observed exclusively in the noise and WBV exposure groups. In conclusion, we demonstrated that, as in exposed workers, prolonged exposure to the combination of LF noise and WBV determines an increase in SCE level in mice while LF noise alone is not effective in SCE induction.