Mutagenesis vol. 19 no. 4 pp. 299-305,
July 2004
© 2004 UK Environmental Mutagen Society/Oxford University Press
Transmission of 
-ray-induced unstable chromosomal aberrations through successive mitotic divisions in human lymphocytes in vitro
Genetic Toxicology and Chromosome Studies Section, Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400 085, India
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Abstract |
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Transmission of unstable chromosomal aberrations through successive mitotic divisions M1–M4 was analysed in 3 Gy
-ray-irradiated G0 human lymphocytes in vitro, harvested at 50, 72 and 96 h. Bromodeoxyuridine (BrdU) incorporation and subsequently the fluorescence plus Giemsa (FPG) method allowed the cell cycle status of each cell scored to be ascertained. Higher dicentric frequencies were observed in cells within the same post-irradiation division derived from extended culture times, indicating either a delay in cell cycle progression of aberrant cells or different lymphocyte sub-populations. The yield of complete dicentrics decreased by a factor of two in passing from M1 to M2 and showed further reductions of 26 and 44%, respectively, in moving from M2 to M3 and from M3 to M4. In the case of conventionally stained metaphases, wherein the cell cycle status does not enter the picture, the dicentric frequencies showed a reduction in yield of 39.6% at 72 h and 52.1% at 96 h compared with 50 h, because the cells analysed comprise a mixture of metaphases in different cell cycles. In the FPG stained first division metaphases, 92–100% of dicentrics analysed at 50, 72 and 96 h and in the conventionally stained metaphases, 90–94, 72–84 and 54–80% of dicentrics analysed at 50, 72 and 96 h respectively were complete dicentrics (with fragments). In the M1, M2, M3 and M4 metaphases analysed, 92–100, 50–89, 20–70 and 10–50% of dicentrics, respectively, were complete dicentrics and 55–75, 53–68, 43–57 and 36–50% excess acentrics, respectively, were seen in cells with complete dicentrics. Interindividual variation was observed in cell cycle kinetics, radiation-induced chromosomal aberrations and micronucleus frequencies. A salient feature of the delayed effects was the induction of giant cells and the mirror dicentrics observed in them. The reduction in dicentric frequencies due to either cell cycle delay or cell death in successive generations is aided by the multiplicity of aberrations. Bridge–breakage–fusion (BBF) events mediated by dicentric chromosomes may also be instrumental in the perpetuation of chromosomal instability. |
Introduction |
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Genomic instability is, in general, a principal characteristic of virtually all neoplastic cells (Coleman and Tsongalis, 1999
; Vessey et al., 1999; Hendry, 2001). Experimental confirmation gave credence to the fact that ionizing radiation can cause genomic instability (Mendonca et al., 1993; Morgan et al., 1996; Murnane, 1996; Little et al., 1997; Little, 1999). That ionizing radiation can induce delayed chromosomal instability is evident from a number of studies in recent years (Lambert et al., 1998; Holmberg et al., 1998a,b,c; Kaplan and Morgan, 1998; Boyle et al., 2002). Chromosome instability can be manifested as an increased frequency of chromosome aberrations within a clonal or non-clonal population of cells occurring multiple generations after exposure to both low (X-rays and -rays) and high LET radiation (
particles) and also single particle traversal (Minkler, 1971; Kadhim, 1992, 1994, 1995; Holmberg et al., 1993; Marder and Morgan, 1993; Martins et al., 1993).
The present study reports on the transmission frequencies of unstable chromosomal aberrations induced by -rays in human lymphocytes in vitro in four subsequent cell divisions. Extended lymphocyte culture times from 50 to 72 and 96 h, 5-bromo-2'-deoxyuridine (BrdU) incorporation and fluorescence plus Giemsa (FPG) staining enabled chromosomal aberration analysis in successive M1–M4 cell divisions post-irradiation. Although not an ideal surrogate for the in vivo elimination of chromosomal damage over successive generations, data presenting the persistence of aberration yield with respect to cell cycles are still a realistic representation of the in vivo passing of time. Higher dicentric frequencies observed in cells within the same post-irradiation division derived from extended culture times indicate either a delay in cell cycle progression of aberrant cells or different lymphocyte sub-populations. Even for baseline data, the dicentric frequencies analysed exclusively in first cycle metaphases (M1) are of critical importance, since the frequency of unstable aberrations may be underestimated in M2 and later divisions (Stephan and Pressel, 1999). Boei et al. (1996) and Guerrero-Carbajal et al. (1998) found almost constant dicentric yields with later sampling times when the cell cycle parameter was not considered. Pala et al. (2001) stressed the importance of the protocol adopted for scoring dicentrics, i.e. with or without their associated fragments, and how it could affect the way the dicentrics are seen to be transmitted. Less than 40% of the complete dicentrics scored in M2, M3 and M4 cells exhibited a second fragment.
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Materials and methods |
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Heparinized blood samples were obtained from five healthy donors (male, non-smokers, aged 38–45 years), without any history of smoking, tobacco chewing, alcohol consumption or drug taking, for medical or other reasons. Radiation was delivered by a 60Co source at a dose rate of 0.77 Gy/min. A dose of 3 Gy was used for irradiation of blood samples. Irradiated blood samples were kept at 37°C for 1 h before setting up cultures. Phytohaemagglutinin (PHA)-stimulated whole blood cultures, 15 ml each, (Ham’s F10 medium, 200 mM L-glutamine, 10% foetal bovine serum, 0.2 ml reconstituted PHA, 0.5 ml whole blood per 5 ml culture volume, no antibiotics), with or without 10.0 µg/ml BrdU (Sigma), for chromosome aberration analysis and 5 ml cultures for cytochalasin B blocked micronuclei (CBMN) analysis were set up, following routine procedures (Krishnaja and Sharma, 1994
). A 5 ml culture was harvested at each time point (50, 72 and 96 h). Cultures for chromosome analysis were treated with 0.02 µg/ml colcemid 3 h before harvest. Air dried preparations of hypotonically treated, methanol:acetic acid fixed lymphocytes were made using routine techniques for chromosome analysis. Cells incorporating BrdU were stained by a modified FPG technique (Perry and Wolff, 1974; Krishnaja and Sharma, 1998). Chromosome preparations which had been aged for 2 days were stained with Hoechst 33258 (100 µg/ml in distilled water) for 20 min, rinsed in tap water, mounted in Sorensen’s buffer (M/15, pH 8.0 adjusted with 5% NaOH) under a coverslip and were exposed to 360 nm light from a black lamp (distance 2 cm, 20 J/m2/s) for 12 min on a slide warming tray at 60°C. Finally, slides rinsed in ice-cold Sorensen’s buffer, pH 6.8, followed by tap water were stained with 4% Giemsa (Merck) in Sorensen’s buffer, pH 6.8. The optimum staining time varied between slide and stain batches, but was of the order of 
8 min (Krishnaja and Sharma, 2003). Harlequin staining of BrdU-substituted chromatids by FPG staining allowed discriminative scoring of chromosomal aberrations in the first, second, third, fourth and fifth mitoses after irradiation.
Conventionally stained and FPG stained first, second or subsequent division metaphase spreads from G0 irradiated lymphocytes, sampled at 50, 72 or 96 h, were examined for unstable chromosome aberrations such as dicentrics and multiple forms, centric rings each with fragments and excess acentrics (interstitial and terminal deletions) consisting of fragments, minutes and acentric rings (Lloyd et al., 1987). Each metaphase had to contain 46 centromeres. The dicentrics and rings seen without accompanying fragments were scored separately. The sister chromatid exchanges (SCEs) were also analysed in 50 second division metaphases, presented as frequency of SCEs per cell and the proliferation rate index (PRI) was evaluated in 200 metaphases by scoring the number of cells in the first, second, third or subsequent divisions in the FPG stained slides (Lambert et.al., 1983; Krishnaja and Sharma, 2003).
In the CBMN assay, 6 µg/ml cytochalasin B was added to the cultures 24 h post-initiation as described earlier and cells were harvested at 72 h. Following a 5 min 0.8% cold KCl treatment and standard fixation, including 1% formaldehyde in the second fixative, cells were stained with 1% Giemsa (Merck) in Sorensen’s buffer, pH 6.8, for 20 min. Total numbers of micronucleated cells (MNBN) and total number of micronuclei (MN) were determined in 1000 binucleated cells with well preserved cytoplasm according to established criteria. The nuclear division index (NDI) was determined by scoring the number of mononucleate, binucleate, trinucleate, tetranucleate and more (polynucleate) cells in 1000 viable cells as previously described. (Fenech, 1993; Krishnaja and Sharma, 1994; Krishnaja and Sharma, 1998).
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Results |
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Chromosomal aberrations in human peripheral blood lymphocytes, grown in the presence of BrdU, collected at 50, 72 or 96 h were analysed in the first four post-irradiation (0 and 3 Gy) divisions (Figure 1). Chromosomal aberrations were also analysed in irradiated human lymphocytes grown without BrdU, collected at 50, 72 or 96 h. Since the culture time of in vitro irradiated lymphocytes here has been extended to 96 h, the data for the irradiated cultures are divided by cell cycle and sampling times as well. Compared with the conventional staining at 50 h, there were increases of 12.5, 31.25 and 56.25% dicentric frequencies, respectively, in first cycle cells analysed in BrdU cultures at 50, 72 and 96 h. The increase in dicentric frequencies in first division metaphases in individual samples ranged from 1.79 to 25% at 50 h, 10.7 to 59% at 72 h and 35 to 107% at 96 h. This may be due to the differential contribution of aberrant cells entering mitosis late depending on the average generation time of the donor studied. The BrdU-labelled metaphases analysed revealed a high variability of cell cycle duration among the lymphocytes of five donors. A delay in cell cycle progression of chromosomally aberrant cells is evident from the higher dicentric frequencies seen in cells within the same post-irradiation division, when derived from later sampling times (Figure 1). A higher dicentrics per cell frequency was noted in the combined FPG stained first division metaphases collected at 50, 72 and 96 h, compared with 50 h FPG stained first division metaphases, as well as the conventionally stained metaphases analysed at 50 h, in all the donors (Figure 2).
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In general, the aberration frequencies for both dicentrics and excess acentric fragments were higher in cells derived from later sampling times for cells arrested within the same mitosis. For example, in the first mitosis, the dicentrics per cell frequencies increased from 0.34–0.68 at 50 h to 0.46–0.71 at 72 h and 0.62–0.82 at 96 h (Table I). All types of aberrations showed a reduced frequency between the first and subsequent mitoses after irradiation. The frequencies of dicentrics progressively decreased from 0.46–0.71 in the first to 0.22–0.30 in the second and 0.06–0.24 in the third division mitoses in 72 h cultures. In 96 h cultures, the dicentric frequencies again showed reduction from 0.62–0.82 in the first to 0.25–0.56 in the second, 0.18–0.58 in the third, 0.11–0.41 in the fourth and 0.03–0.10 in the fifth division mitoses. The dicentric frequencies showed a reduction of about 48–57% between the first and second mitosis, 57–73% between the first and third and 76% between the first and fourth mitosis. The total dicentric frequencies showed a 26% reduction from M2 to M3 and 44% reduction from M3 to M4. Dicentrics were reduced by 40.8% in M2, 32.6% in M3, 49.2% in M4 and 75.8% in M5, compared with each preceding divisions at the 96 h sampling time. Differential responses of individual donors were found in the dicentric frequency reductions from the M1 to M5 divisions.
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In the case of conventionally stained metaphases without BrdU incorporation, the dicentric frequencies showed a reduction of 39.6% at 72 h and 52.1% at 96 h (Table II). When the total dicentric yields irrespective of the cell cycle number are plotted against extended culture time, the reduction in overall dicentric yield is marked, due to the fact that analysable cells often comprise a mixture of metaphases in different cell cycles at each sampling time. In the first division metaphases sampled at 50, 72 and 96 h, 92–100% of dicentrics analysed and in the conventionally stained metaphases, 90–94% of dicentrics analysed at 50 h were complete dicentrics. Conventionally stained cells showed 72–84 and 54–80% complete dicentrics at 72 and 96 h respectively. In the second, third and fourth division FPG stained metaphases, 50–89, 20–70 and 10–50% of dicentrics were complete dicentrics. In the present data only 39, 24 and 33% of the complete dicentrics scored in M2, M3 and M4, respectively, exhibited a second fragment. The dicentric yield at 96 h appears to plateau from M3 to M4. The frequency of rings was too small to allow a separate analysis.
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A salient feature of the delayed effects was the induction of giant cells and the mirror dicentrics observed in metaphases M2–M4 analysed at 72 and 96 h (Figure 3A and B). The absolute number of giant cells represented <2% of the total number of metaphases scored at 72 and 96 h. No dicentric chromosomes were found in the 100 cells each analysed from the non-irradiated control cultures from the five donors. The frequencies of chromosome and chromatid breaks were found to be
2% in these control cultures. The data derived from control cultures did not show any effect of cell cycle on the aberration frequencies.
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Losses of excess acentrics from the first to second cell cycle at 50, 72 and 96 h were 23.1–34.6, 47.5–62.9 and 37.0–86.5%, respectively. From the second to third cell cycle at 72 and 96 h, 50–69.2 and 41.2–75% excess acentrics were lost. A 21.4–63.6% reduction and, in two cases, 1.5- to 4.6-fold increases from the third to fourth division were seen at 96 h (Table III). An 84–88.2% reduction in excess acentrics was observed from the fourth to fifth divisions. In conventionally stained metaphase spreads without BrdU incorporation, 13.3–67.9 and 47.4–77.8% reductions in excess acentric fragments were observed from 50 to 72 and from 72 to 96 h, respectively. In one donor a 16.7% increase in excess acentric fragments from 72 to 96 h was observed. In the FPG stained cells, in two cases 36.5 and 85.7% increases in excess acentrics were seen between 50 and 96 h cycle I metaphases.
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SCE frequencies in 3 Gy irradiated cells ranged from 4.42 to 6.48 with a mean of 5.63 and, in controls from 4.28 to 6.34 with a mean of 5.38. Using BrdU incorporation it was seen that irradiation slightly slowed down the cell cycle, as is evident from the PRI values at 72 and 96 h. PRI values in 3 Gy irradiated cells ranged from 1.69 to 1.94 with a mean of 1.79, whereas in controls the PRI ranged from 1.83 to 2.54 with a mean of 2.2 at 72 h. The PRI values ranged from 2.28 to 3.18 in 3 Gy irradiated cells, compared with 2.53–3.23 in controls at 96 h. The cell cycle delay was evident from the higher number of cycle I cells and fewer cycle IV cells in 3 Gy irradiated cells compared with controls.
At 3 Gy MN frequencies ranged from 0.42 to 0.67 per cell with a mean of 0.56, whereas MNBN ranged from 0.31 to 0.47 with a mean of 0.40 and in 0 Gy MN ranged from 0.004 to 0.010 with a mean of 0.007 and MNBN ranged from 0.004 to 0.009 with a mean of 0.006. The NDI in 3 Gy irradiated cells ranged from 1.32 to 1.78 with a mean of 1.55, whereas in controls the NDI ranged from 1.84 to 2.07 with a mean of 1.96. In a single sample analysed at 50, 72 and 96 h the MN frequencies per cell observed were 0.27–0.28, 0.42–0.67 and 0.30–0.37, respectively. At 50 h in 3 Gy irradiated lymphocytes, 10–20% of the cells were binucleate and 76–90% remained mononucleate. In irradiated cells at 72 and 96 h, 57–71 and 49–50% cells, respectively, remained mononucleate. An increase in binucleate cells of 44–47% was noticed in 3 Gy irradiated lymphocytes at 96 h, compared with 25–36% at 72 h. Normally, at 96 h many early dividing unirradiated lymphocytes do not remain binucleate but become multinucleate (three or more nuclei) due to an attempted second division during this time frame. Consequently, the accumulation of multinucleated cells lowers the frequency of binucleate cells. In contrast, due to the cell cycle delay, many of the 3 Gy irradiated late dividing mononucleate cells became binucleate during this time frame, with a consequent increase in the number of binucleated cells at 96 h. Interindividual variation was observed in cell cycle kinetics, chromosomal aberrations and MN frequencies.
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Discussion |
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Beginning with the demonstration that a single
particle is able to induce chromosomal instability in primary human T lymphocytes, ionizing radiation-induced chromosome instability has been found to occur in a variety of cell types. (Kadhim et al., 1992; Holmberg et al., 1993; Marder and Morgan, 1993; Grosovsky, 1996). The frequency of dicentric chromosomes declines during the first few divisions following irradiation (Martins et al., 1993). There is substantial evidence that BBF cycles are a major mechanism of forming novel dicentrics and other chromosomal rearrangements (Grosovsky, 1996; Morgan et al., 1996). The present study reports on the asymmetric aberration frequencies observed post-irradiation in M1–M4 human lymphocyte metaphases. Extended lymphocyte culture times from 50 to 72 and 96 h combined with FPG staining and cell cycle analysis in lymphocytes from five donors, to simulate the elimination of chromosomal damage that occurs over time in vivo following successive cell divisions, allowed more representative aberration scores which reflect the radiosensitivity of a large part of the cell cycle compared with the yield obtained from a single fixation time. In this case loss of aberrations is mainly a function of the transmissibility of aberrations through successive cell divisions into viable daughter cells.
The yield of complete dicentrics decreased by more than a factor of two in going from M1 to M2. The possibility of still scoring dicentrics that had lost their associated fragments as dicentrics with fragments in M2 exists, because of the initial excess fragments present in M1. Hence, in an M2 cell, a complete dicentric should always be accompanied by a second excess acentric fragment. It is also possible that acentric fragments in M1 may form a micronucleus, which may not always cycle in synchrony with the main nucleus and may not contribute two fragments to M2 metaphases. Hence, a large number of acentrics in M1 cells do not produce paired acentrics in M2 cells. Thus the decline in complete dicentric yield, by a factor of two or more in going from M1 to M2, would also be dependent on the dose. The total dicentric yield in M2 was more than 40% of the equivalent yield in M1 at 50, 72 and 96 h. Most dicentrics and mirror dicentrics observed in the giant cells were accompanied by fragments. Since the number of excess fragments was high, a good estimate of the fraction lost cannot be obtained from the frequency of dicentrics without fragments. The results point to random segregation of acentrics, >30% being eliminated in each generation.
The reduction in overall dicentric yield is marked when the total dicentric yields are plotted against extended culture time, without taking account of the cell cycle number. Increasing numbers of M2–M5 cells over M1 cells at these extended culture times result in low dicentric yields by conventional staining at 72 and 96 h. The yield of dicentrics in M1 cells at 96 h may be different from the yield in early cycling lymphocytes, because of the presence of a subset that reached M1 later having an increased frequency of dicentrics. Even at this extended culture time a large number of aberrant M1 cells that arrived late in cell division are still present. This could possibly represent a set of B lymphocytes entering first mitosis somewhat later than T cells (Guerrero-Carbajal et al., 1998). In PHA-stimulated cultures, dependency of the B lymphocyte blastogenic response on T lymphocytes has already been shown (Han and Daday, 1978) and earlier studies have found no significant difference in chromosomal aberration frequencies between irradiated human T and B cells (Schwartz and Gaulden, 1980). The centric rings with fragments scored comprised 5 and 10% of the dicentric yields in M1 and M2 cells, respectively, suggesting that centric rings are better able to negotiate mitosis compared with dicentrics. The persistence of centric rings in later divisions is probably due to the fact that rings are less likely to form a lethal anaphase bridge at cell division, unlike many dicentrics. However, the frequency of rings is too small to allow a valuable analysis.
Compatible with a 50% reduction per generation, the dicentric frequencies show a progressive decrease in relation to the number of cell divisions after irradiation. This decrease is more regular in 72 h than in 96 h cultures. Higher dicentric frequencies were seen in 96 h than in 72h cultures for the equivalent number of cell cycles. Radiation-induced chromosome rearrangements may influence cell survival and duration of the cell cycle. In certain individuals a drastic reduction in aberration frequencies between the second and third divisions was noticed. This might be due to an increased generation time for aberrant cells, which consequently would not have reached their third cell division.
Earlier studies pointed out that cell survival rate from the first to second division is mainly influenced by two-hit aberrations and loss of fragments is only of secondary importance (Sasaki and Norman 1967; Carrano and Heddle, 1973). Sasaki and Norman (1967) have further estimated that a dicentric chromosome in human lymphocyte culture will survive either the first or the second division in vitro with a probability close to 0.5 and that acentric fragments are distributed to daughter cells with a probability of 0.3. Kovacs et al. (1994) and Boei et al. (1996) have also suggested 50% elimination of asymmetrical chromosome exchanges at each cell generation, although strictly not applicable across all cell cycles. Anaphase bridging of these two-hit aberrations influences the cell survival rate from the first to second generation (Bauchinger et al., 1986).
Our results are in agreement with Tanzarella et al. (1986) and Boei et al. (1996), who presented evidence for increased frequencies of aberrations in lymphocytes which arrive late in mitosis. Lymphocyte cultures contain a heterogeneous population of cells with different generation times and damaged cells might be affected in their progression rate. Anderson et al. (2000) reported that the frequency of exchange aberrations in first division lymphocytes increased significantly with sampling time after exposure to 0.5 Gy particles but not after 3 Gy X-rays. George et al. (2001) also pointed out that prolonged cell cycle delays could lead to an underestimation of high LET-induced damage if metaphase cells are analysed at one time point when the majority of the cell population reaches first division in culture.
The involvement of BBF in genetic instability induced by ionizing radiation has been proposed previously (Marder and Morgan, 1993). Prolonged genetic instability after treatment with ionizing radiation could also be sustained by a chain reaction of chromosome rearrangements (Murnane, 1996). While the target for radiation-induced chromosomal instability is the nucleus, DNA double-strand breaks seem necessary but not sufficient for the induction of chromosomal instability (Kaplan and Morgan, 1998). However, the target for radiation-induced chromosomal instability is distinct from the target for cell killing and chromosomal rearrangements. Restriction enzymes, when electroporated into cells, cause DNA double-strand breaks, cell killing and chromosomal rearrangements, yet do not induce chromosomal instability (Limoli et al., 1997).
Giant cells containing multiple aberrations induced in irradiated cultures at 72 and 96 h represented <2% of the total number of metaphases scored. None of the unirradiated metaphases examined showed any giant cells. The occurrence of second division replicated dicentrics in both giant cells indicates that chromosomal aberrations were formed during the period preceding the last round of replication and both rounds of replication took place in vitro. Replication may be accompanied by blocked karyokinesis and formation of a tetraploid cell. Some of the aberrations seemed to be formed during the last mitotic division cycle since no replicated chromosome aberrations were seen in these giant cells. The cellular mechanisms that lead to the formation of giant cells containing multiple complex rearrangements are unknown, but may reflect the delayed effects of irradiation on the mitotic spindle apparatus or faithful segregation of chromatids at cytokinesis (Cross et al., 1995; Fukasawa et al., 1996; Morgan et al., 1996). X-irradiation has been shown to interfere with the highly ordered sequence of events leading to cytokinesis in Saccharomyces uvarum, which affects cell cycle progression and hence causes giant cell formation (Baumstark-Khan et al., 1986). Spontaneous premature condensation, probably due to radiation-induced cell cycle delays in the late S and G2 phases and enhanced intracellular levels of cyclin B1 may play a role in the mechanism underlying radiation-induced giant cells (Ianzini and Mackey, 1997).
Since all chromosomal damage that survives the first post-irradiation division, after subsequent replication results in chromosome type aberrations, the primary origin of chromosome-type aberrations found in second or later generation metaphases cannot be unequivocally identified. Mitotic cells are generally collected at one time point. However, any perturbation of cell cycle progression in cells damaged by radiation exposure will influence the frequency of aberrations at an early collection point. Severely damaged cells could experience more prolonged cell cycle delays and extended culture times can take into account the complete timecourse of aberrations for a meaningful quantification of chromosomal damage.
The loss of dicentrics is not only related to their poor transmissibility but also to the presence of other chromosomal rearrangements. In essence, it is the multiplicity of aberrations which determines the reduction in dicentric frequencies, which results both from cell death as well as slowing down of the cell cycle. Premature chromosome condensation, however, could avoid problems connected with complicated cell cycle delays. It is well known that radiation exposure causes cells to arrest in G1 in order to elicit apoptosis or induce repair of damage before S phase and mitosis (Liu et al., 1995). BBF events mediated by dicentric chromosomes may also be instrumental in the perpetuation of chromosome instability. These observations may have important implications for mechanistic studies of radiation effects and risk assessment of radiation exposure.
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1To whom correspondence should be addressed. Tel: +91 22 2559 3949; Fax: +91 22 2550 5151; Email address: krishnja{at}apsara.barc.ernet.in
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