Mutagenesis, Vol. 14, No. 4, 357-364,
July 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Analysis of bleomycin-induced chromosomal aberrations in Chinese hamster primary embryonic cells by FISH using arm-specific painting probes
MGC Department of Radiation Genetics and Chemical Mutagenesis, Leiden University Medical Centre, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands
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Abstract |
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Chinese hamster primary embryonic cells (at G1 phase) were treated with 1.0 or 3.0 µg/ml bleomycin and chromosomal aberrations in first division metaphases were analysed by fluorescence in situ hybridization (FISH) using arm-specific painting probes for chromosomes 3, 4, 8 and 9. We observed that bleomycin induced all classes of chromosome-type aberrations very efficiently. The interesting findings were: (i) the frequency of induced interstitial translocations (i.e. insertions) was approximately equal to that of reciprocal translocations; (ii) the frequency of induced pericentric inversions was higher than that of centric rings. In our earlier studies, we found that X-rays induced a low frequency of interstitial translocations in comparison with reciprocal translocations and equal frequencies of centric rings and pericentric inversions. These data suggest that bleomycin differs from X-rays with respect to the induction of some specific types of aberrations. The results of a
2 test examining the hypothesis that formed aberrations among the chromosomes or chromosome arms are randomly distributed on the basis of their relative lengths revealed a differential involvement of these chromosomes in the aberrations following exposure to bleomycin. In general, chromosome 8 was found to be more involved in induced aberrations than expected, chromosome 4 was randomly involved, whereas chromosomes 3 and 9 were less involved. This study demonstrates the utility of arm-specific painting probes for efficient detection of a large variety of chromosomal aberrations induced by bleomycin. |
Introduction |
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Bleomycin is a radiomimetic antitumour agent with potent genotoxic properties, including induction of chromosomal aberrations. The cellular target for chromosomal aberrations induced by bleomycin is DNA. It has been proposed that bleomycin, in the presence of Fe(III), induces double-strand breaks by first attacking a single strand of DNA between a guanine and a pyrimidine. Activated bleomycin is then regenerated and cleaves the opposite DNA strand yielding blunt-ended or single base overhanging double-strand breaks (Povirk et al., 1989
). It has been demonstrated that bleomycin acts as an S phase-independent agent since it induces aberrations at all stages of the cell cycle. Chromosome-type aberrations are induced when cells are treated before DNA replication (i.e. G0 or G1 phase) and chromatid-type aberrations are induced during and after DNA synthesis (i.e. S or G2 phase) (Dresp et al., 1978; Povirk and Austin, 1991). The similarities between bleomycin and ionizing radiation in induction of chromosomal aberrations are the basis for considering bleomycin as a radiomimetic compound. The dose–response relationship for induction of chromosomal aberrations by bleomycin has been found to be linear and the aberrations induced by bleomycin to be overdispersed (Dresp et al., 1978). It has been found that the induction kinetics of chromosomal aberrations by neutrons (high LET radiation) is linear (Lloyd et al., 1976) and distributions of aberrations induced by neutrons tend to be overdispersed (Edwards et al., 1979), whereas aberrations induced by X-rays and 
-rays (low LET radiation) tend to be linear-quadratic (Lloyd et al., 1975) and Poisson distributed (Edwards et al., 1979). Therefore, it has been suggested that the clastogenic effects of bleomycin are similar to those of high LET radiation (Dresp et al., 1978; Hoffmann et al., 1993).
While chromosomal aberrations have been observed following exposure to ionizing radiation as well as chemical agents, the mechanisms underlying the formation of aberrations are not well understood. Many studies have been carried out to investigate the formation of chromosomal aberrations following exposure to ionizing radiation. A large amount of data on ionizing radiation-induced chromosomal aberrations analysed by fluorescence in situ hybridization (FISH) using DNA libraries is now available. The data pertain to species-specific differences, dose–response curves, in vivo and in vitro results, involvement of individual chromosomes, relative frequencies of specific types of aberrations, etc. (Lucas et al., 1989; Natarajan et al., 1992, 1996; Boei et al., 1995; Dominguez et al., 1996; Simpson and Savage, 1996; Tucker et al., 1997; Grigorova et al., 1998). The FISH technique has proved to be useful for cytogenetic analysis of spontaneous as well as induced chromosomal aberrations. However, only a few studies on bleomycin-induced chromosomal aberrations using FISH have been carried out so far (Hoffmann et al., 1994; Ellard et al., 1995).
The present study was designed: (i) to determine the frequencies of bleomycin-induced chromosomal aberrations in Chinese hamster cells detected by FISH using arm-specific probes; (ii) to estimate the relative frequencies of specific types of aberrations induced by bleomycin; (iii) to compare the involvement of individual chromosomes in bleomycin-induced aberrations. Data obtained in this study have proved to be useful for making quantitative and qualitative comparisons between bleomycin- and ionizing radiation-induced chromosomal aberrations.
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Materials and methods |
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Cells and culture conditions
Chinese hamster primary embryonic (CHE) cells from a male embryo were used in the present study. The cells were grown in F10 medium supplemented with 15% fetal calf serum (Gibco BRL, Grand Island, NY) and antibiotics (final concentrations 100 U/ml penicillin and 0.1 mg/ml streptomycin; Sigma, St Louis, MO), at 37°C in a 95% humidified incubator with 5% CO2.
Chemical and treatment
Bleomycin was purchased from Londbeck (Amsterdam, The Netherlands). The drug was dissolved in phosphate-buffered saline (PBS). The cells were grown to a density-inhibited, confluent state and treated for 1 h with bleomycin at final concentrations of 0 (PBS control), 1.0 or 3.0 µg/ml. The choice of doses and the duration of bleomycin treatment were based on results of our earlier studies (Darroudi and Natarajan, 1989). Treated cells were washed carefully with PBS (three times), then subcultured in fresh medium at a dilution ratio of 1:4. 5-Bromodeoxyuridine (BrdU) (5 µM final concentration; Sigma) was added to each culture. Cells were allowed to recover for 18 h under the same conditions as described above.
Metaphase preparations
Metaphases were arrested with colcemid (0.1 µg/ml; Sigma) for 2 h, trypsinized and harvested 20 h after culture initiation. Following hypotonic shock with 0.075 M KCl at 37°C for 20 min, the cells were fixed with methanol and acetic acid (3:1). After two additional changes of fixative, the cell suspension was dropped onto clean slides. The slides were stored at –20°C until in situ hybridization was performed.
Arm-specific probes
Arm-specific painting probes for Chinese hamster chromosomes were generated by microdissection and degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR) (Xiao and Natarajan, 1998). The products from third round PCR amplification of microdissected chromosome fragments were used as probes. Probes for both arms of chromosomes 3, 4, 8 and 9 were used in this study. These probes were directly labelled with digoxigenin-11-dUTP (short arm probes) or biotin-16-dUTP (long arm probes) by PCR.
In situ hybridization
In situ hybridizations were performed as described earlier (Xiao et al., 1996). Briefly, a cocktail of digoxigenin- and biotin-labelled probes (each probe ±100 ng) for the short and long arms of the chromosomes, together with Chinese hamster genomic DNA (2 µg), was diluted in hybridization mix containing 50% formamide, 2x SSC, 10% dextran sulfate, 50 mM phosphate buffer (pH 7.0) to a total volume of 20 µl and denatured on a slide under a 24x50 mm coverslip at 80°C for 3.5 min. Hybridization was performed overnight in a moist chamber at 37°C. Probes for the short arms of the Chinese hamster chromosomes were labelled with digoxigenin and detected by sequential application of mouse anti-digoxigenin, digoxigenin-conjugated sheep anti-mouse and sheep anti-digoxigenin antibodies conjugated with TRITC (Boehringer, Mannheim, Germany), giving red (TRITC) signals. The biotinylated probes for the long arms were detected by two layers of FITC-conjugated avidin and amplified with one layer of anti-avidin antibody (Vector Laboratories, USA), giving green (FITC) signals. Slides were counterstained with 0.15 µg/ml 4',6-diamidino-2-phenylindole (DAPI), staining chromosomes blue, in Vectashield mounting medium (Vector laboratories).
Analysis of aberrations
Analysis of the cell cycle stage of the cultured cells was performed using the fluorescence plus Giemsa technique (Perry and Wolff, 1974). The fraction of cells in the second mitosis was very low for the PBS control (<1%) and no second division cells were observed in bleomycin-treated cells at the time of fixation.
Analysis of FISH slides was performed using a Zeiss microscope equipped with a triple filter to simultaneously visualize the DAPI (blue), FITC (green) and TRITC (red) signals. Colour images were collected using a computer-controlled Zeiss epifluorescence microscope equipped with a cooled charge-coupled device (CCD) camera (Photometrics, USA) and IP Lab Spectrum software (Figure 1A–H).
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All cells in metaphases were scored, except for those which were polyploid or had lost the painted chromosome(s). Aberrations involving the painted chromosomes were analysed. Attention was paid to identifying aberrations involving individual chromosomes as well as individual painted arms. Classification of aberrations was performed using a combination of the conventional (Savage, 1976
), S&S (Savage and Simpson, 1994a,b) and PAINT (protocol for aberration identification and nomenclature terminology; Tucker et al., 1995) nomenclature systems. The conventional classification has been widely used for the study of induced aberrations in solid stained, banded and painted chromosomes. The S&S and PAINT systems have been proposed for the study of induced aberrations in painted chromosomes. The S&S system was designed to unravel the fundamental mechanism underlying the formation of ionizing radiation-induced chromosomal aberrations. It can only be applied to single chromosome paints (or multicolour cocktails) in combination with a centromeric probe and unique FISH patterns are taken into account. The PAINT system has been proposed by a group of cytogeneticists (Tucker et al., 1995). The system describes the aberrations involving the painted chromosomes without any consideration of the mechanism involved. Savage and Tucker (1996) have noted that the PAINT and S&S systems are designed for different purposes and are not mutually exclusive but complementary. In practice, we combined the three systems and classify all aberrations as follows.
Aberrant chromosomes.
Only painted chromosomes involved in aberrations were counted. A painted chromosome (including both arms) involved in any kind of aberration was recorded as one aberrant chromosome. If homologous chromosomes were simultaneously aberrant, two aberrant chromosomes were recorded. The frequencies of aberrant chromosomes give an overall impression of the extent of chromosome damage.
Breakages
. Breakages of painted chromosomes or chromosome arms were scored depending on the number of breaks of the painted chromosomes or arms, painted fragments and exchanges detected by FISH. For example, if only a single painted fragment was found, we counted it as one breakage in that painted arm. We also counted it as one breakage in a painted arm if the painted arm was involved in an apparently simple dicentric or translocation (see below). Two breakages were counted if two identical colour fragments were observed, and so on. If a fragment was composed of both painted colours, two breakages (one in each arm) in the painted chromosome were recorded. A painted arm containing an unpainted insertion was counted as having one breakage, but when a painted segment was inserted into a non-painted arm, the painted arm of origin was counted as having two breakages. When the painted chromosome was involved in a simple centric ring or a complete (or simple incomplete) pericentric inversion, two breakages (one breakage of each arm) were recorded. The number of breakages in the painted chromosomes or arms involved in complex exchanges was also determined according to the FISH patterns. For example, the complex 2G (S&S nomenclature), which contains a dicentric and a terminal translocation, was counted as having one breakage in the painted arm involved. In complexes 3A–3D and 3H–3M (Savage and Simpson, 1994b) the painted arms involved were recorded as having two breakages. Three breakages were counted in the arm which was involved in the complex 3E.
According to the breakage–reunion theory (Sax, 1940; Lea, 1946; reviewed in Savage, 1996), initial (chromosome) breaks induced by ionizing radiation have one of three fates: (i) to reconstitute, i.e. the two ends join back as they were, restoring the original configuration; (ii) to rejoin illegitimately with other breaks, forming an exchange; (iii) to remain open as a fragment seen in metaphase. Although reconstituted breaks cannot be detected, the opened and the illegitimately rejoined breaks can be detected in the first metaphase following aberration induction. Therefore, scoring either fragments or exchanges alone may underestimate the extent of induced chromosomal damage. The breakage of painted chromosomes or chromosome arms scored in the present study is based on the origin of painted fragments and exchanges detected by the FISH technique. The number of breakages detected by FISH in first division cells does not represent all of the breaks that occurred immediately after exposure, but the residual breaks at the time point examined, including unjoined breaks (e.g. fragments) as well as rejoined breaks (e.g. exchanges). The total number of breakages of painted chromosomes or chromosome arms may thus be a useful indicator for quantifying chromosomal aberrations induced by ionizing radiation and chemical agents. It may also be useful for exploring the mechanism(s) of the formation of chromosomal aberrations.
Colour junctions
. Colour junction defines the conjunction of two different colours, where exchanges occur. Colour junctions counted included those between a painted arm and an unpainted chromosome (p–unpainted and q–unpainted) as well as between two painted arms (p–q). A reciprocal dicentric (i.e. a dicentric accompanied by a bicolour acentric fragment) or a reciprocal translocation was counted as having two colour junctions. A dicentric accompanied by a single colour fragment (for example Figure 1A
) or without a fragment was counted as having one colour junction. An insertion was counted as having two colour junctions.
The use of colour junctions to quantify induced chromosomal exchanges was first recommended in the PAINT nomenclature (Tucker et al., 1995). Since any exchange between a painted chromosome and an unpainted chromosome is counted as a colour junction, it is useful as an overall estimate of total exchanges induced by the agents tested. However, it should be noted that the number of colour junctions is not comparable with any particular type of exchange because both a complete simple (e.g. reciprocal dicentric or translocation) and a complex exchange (e.g. interstitial translocations or a complex containing one dicentric and one terminal exchange) have two colour junctions, whereas apparently simple exchanges, such as a dicentric with a bicolour fragment and a dicentric with two separate fragments, have different numbers of colour junctions.
Dicentrics
. Apparently simple dicentrics were those in which one arm of a painted chromosome joined with another single centric (painted or unpainted) chromosome fragment. This type of aberration is usually accompanied by an acentric fragment of the same colour as the aberrant arm or a bicolour fragment which has the same colours as the dicentric. In the present study, the total numbers of dicentrics included those which were involved in complex exchanges.
Translocations
. Apparently simple translocations were confined to reciprocal translocations and simple terminal translocations (i.e. one way exchanges). A reciprocal translocation was counted as one apparently simple translocation. Simple terminal translocations, i.e. only one terminal translocation involving a painted chromosome (or a painted chromosome arm), included: (i) an arm of a painted chromosome with an unpainted terminal end and accompanied by an acentric fragment painted in the same colour as the arm; (ii) an unpainted chromosome with a painted terminal end; (iii) an arm of a painted chromosome with an unpainted terminal end. Each of them was counted as one terminal translocation. Interstitial translocations (i.e. insertions) included both a painted fragment inserted into an unpainted chromosome and an unpainted fragment inserted into a painted chromosome. Each insertion was counted as one interstitial translocation. Total numbers of translocations included all types of translocations encountered as well as those derived from complex exchanges
Centric rings
. Simple centric rings included those with or without identical painted bicolour or single colour fragments. If an unpainted chromosome(s) or fragment(s) was involved in a centric ring or its fragment(s), the centric ring was classified as a complex one.
Pericentric inversions
. Complete pericentric inversions were defined as chromosomes in which both arms were reciprocally inverted. Incomplete simple pericentric inversions were those in which only one arm was inverted, usually accompanied by a painted fragment. If an unpainted chromosome(s) or fragment(s) was involved in it, this pericentric inversion was counted as a complex one.
Homologous exchanges
. Homologous exchanges were those that occurred between two painted homologous chromosomes. The resolution of the FISH technique is limited in its ability to distinguish reciprocal exchanges between homologous arms, although obvious alterations in their size can be recognized.
Complex exchanges
. Complex exchanges were defined as those consisting of three or more breakages in two or more chromosomes (Savage and Simpson, 1994b). In this study, complex exchanges were scored by splitting them up into individual types of exchanges. For example, in Figure 1E, the number of exchanges in the complex (c,t,f) was determined and recorded as two dicentrics (one involving p and one involving q) and one (p arm) terminal translocation.
Statistics
A standard 2 test was performed to examine the hypothesis that the chromosomal aberrations detected are randomly distributed among the individual chromosomes or their arms on the basis of their size. The expected number of each type of aberration was estimated on the basis of the relative length of chromosomes or chromosome arms, except for apparently simple exchanges. The expected number of apparently simple exchanges was estimated on the basis of the relative corrected length of the chromosomes or the arms (RCL; Savage and Papworth, 1982) for interchanges.
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Results |
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Examples of various types of chromosomal aberrations detected by FISH using arm-specific probes are presented in Figure 1
. Data on the frequencies of bleomycin-induced chromosomal aberrations are presented in Tables I–III.
Frequencies of aberrant chromosomes, breakages and colour junctions
The frequencies of spontaneous aberrant chromosomes, breakages and colour junctions involving the painted chromosomes were similar, indicating that each aberrant chromosome has, on average, one breakage and one colour junction (Table I). These frequencies were increased by bleomycin in a dose-dependent manner. The number of simultaneous aberrations of homologous chromosomes also increased with dose (Table I, column 4). The number of breakages per aberrant chromosome was 1.1–1.4 for the 1 µg/ml group and 1.4–1.8 for the 3 µg/ml group. Most of the induced colour junctions were between painted arms and unpainted chromosomes, the number of p–q colour junctions in the chromosomes studied was between 2 and 10% of total colour junctions.
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The frequencies of induced aberrant chromosomes, breakages and colour junctions were similar for chromosomes 3 and 4. However, these frequencies for chromosome 8 were about twice as high as those for chromosome 9. The results of the
2 test examining involvement of individual chromosomes in aberrant chromosomes, breakages and colour junctions indicate a differential involvement of individual painted chromosomes in aberrant chromosomes, breakages and colour junctions following exposure to 1 and 3 µg/ml bleomycin (Table I, 21 and 23). In most cases, chromosome 8 was more involved than expected and chromosome 4 was involved to the extent expected. Chromosome 3 was less involved than expected in the 1 µg/ml group but proportionally involved in the 3 µg/ml group. Chromosome 9 was proportionally involved in the 1 µg/ml group whereas less involved than expected in the 3 µg/ml group. The results of the 2 test examining involvement of arms in the aberrations (Table I, columns 8 and 13) indicated that the p arm of chromosome 4 and the q arm of chromosome 8 were more frequently involved in colour junctions than expected in the 3 µg/ml group.
Frequencies of dicentrics and translocations
The frequencies of dicentrics and translocations are presented in columns 2 and 3 of Table II. The frequencies of spontaneous dicentrics and translocations were similar, i.e. the ratio of translocations to dicentrics (T/D ratio) in non-treated cells was ~1. The increase in the frequencies of dicentrics and translocations was dose dependent. While the total number of induced translocations was more than the total number of dicentrics, the T/D ratio of apparently simple translocations and dicentrics was ~1 on average. The number of apparently simple dicentrics and translocations was ~30–50% of total dicentrics and translocations (Table II, columns 2–4 and 8), indicating that bleomycin induces a lot of complex exchanges.
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The results of the
2 test indicated that in the 3 µg/ml group (Table II, 23) the total number of dicentrics involving chromosome 3 was less than expected and the total number of translocations involving chromosome 9 was less than expected. Chromosome 4 was proportionally involved in dicentrics and translocations, whereas chromosome 8 was more involved in both types of exchange than expected (Table II, columns 2 and 3). A differential involvement of chromosome arms in dicentrics was also observed in the 3 µg/ml group. The q arm of chromosome 9 was more involved in apparently simple dicentrics than expected (Table II, columns 5–7).
Most of the bleomycin-induced translocations were terminal (Table II, column 3). About 50% of terminal translocations were members of complex exchanges (unpublished data). We found that the number of interstitial translocations induced by 3.0 µg/ml bleomycin was almost equal to the number of induced reciprocal translocations in the majority of cases (Table II, column 3).
Frequencies of centric rings, pericentric inversions and homologous exchanges
The frequencies of centric rings, pericentric inversions and homologous exchanges were increased by bleomycin. These types of exchanges were found to be proportionally distributed among the painted chromosomes, although a high frequency of pericentric inversions involving chromosome 3 was observed in the 3 µg/ml group (Table III, column 5). The frequency of pericentric inversions was higher than that of centric rings, especially for chromosomes 3 and 4. Most of the induced pericentric inversions were either incomplete ones or parts of complex exchanges. In the present study, a typical centric ring (i.e. a centric ring accompanied by a bicolour fragment) or a complete pericentric inversion has two colour junctions. Therefore, it is expected that the number of p–q colour junctions should be about twice the total of centric rings plus pericentric inversions. However, the number of p–q colour junctions observed in the chromosomes studied was less than expected, except for chromosome 4, in which the number of p–q colour junctions was about twice the total number of centric rings and pericentric inversions (Table I, column 10 and Table III, columns 2 and 5). Compared with other types of aberrations, the frequency of bleomycin-induced homologous exchanges was relatively low. One reason for this may be the inability to detect reciprocal exchanges between arms which are painted in identical colours, as most homologous exchanges observed are p–p or q–q changes.
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Discussion |
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Quantifying bleomycin-induced chromosomal damage
To quantify bleomycin-induced chromosomal damage, in addition to different types of chromosomal exchanges (e.g. dicentrics, translocations, rings, etc.) which are routinely used in studies employing the FISH technique, the number of aberrant chromosomes, breakages and colour junctions were taken into account. The frequencies of these different parameters may provide useful information on the induction and formation of different types of chromosomal aberrations. For example, comparing the number of aberrant chromosomes, breakages and colour junctions, one can quickly estimate the degree of total visible chromosome damage induced and the formation of exchanges.
As pointed out in Materials and methods, the number of colour junctions is not comparable with any particular type of exchange. We found that p–q colour junctions were less than twice of the total number of intrachanges, i.e. centric rings and pericentric inversions, indicating that p–q colour junctions cannot correctly reflect the total number of centric rings and pericentric inversions. An obvious reason for this is that most intrachanges observed were incomplete (Figure 1G) and complex (Figure 1B and D), which usually have fewer p–q colour junctions (Table III, columns 2–8 and Figure 1B, D, G and H).
Comparison of chromosomal aberrations induced by bleomycin and ionizing radiation
Chromosomal aberrations induced by bleomycin have been evaluated in different types of cells and compared with those induced by X-rays using a conventional staining method. In CHO-K1 cells, similar frequencies (27 and 29%) of aberrations (including chromatid- and chromosome-type aberration) were induced by 1.2 µg/ml bleomycin and 1 Gy X-rays (Darroudi and Natarajan, 1987, 1989). In lymphocytes of the Indian muntjac, similar frequencies of chromosomal deletions and rearrangements were induced by 40 µg/ml bleomycin and 2 Gy X-rays (Chatterjee and Jacob-Raman, 1988). In peripheral blood lymphocytes of human and rat, similar frequencies of asymmetric exchanges were induced by 80 µg/ml bleomycin and 3 Gy X-rays, while in mouse, 80 µg/ml bleomycin induced a higher frequency of asymmetrical exchanges than that induced by 3 Gy X-rays (Kligerman et al., 1992). It is difficult to make a general conclusion based on these studies, as the species employed, the target cells, the conditions of treatment and the aberrations analysed are different.
Chromosomal aberrations induced by different concentrations (1, 5, 10, 50 and 100 µg/ml) of bleomycin in human lymphocytes (G1) were studied using conventional staining and the FISH technique by Ellard et al. (1995). They demonstrated that painting of only three pairs of chromosomes allowed the detection of similar or even greater numbers of exchanges than conventional staining, implying that bleomycin-induced chromosomal aberrations may be underestimated by conventional cytogenetic methods (Ellard et al., 1995). So far, no report is available on bleomycin-induced aberrations in the cells of other species using the FISH technique.
In the present study, the FISH technique using arm-specific probes was employed. The results obtained demonstrate that bleomycin is an efficient inducer of chromosomal aberrations in Chinese hamster cells. Compared with our data from X-ray experiments (Xiao and Natarajan, 1999), in which interstitial translocations formed only a small proportion, a higher frequency of interstitial translocations was induced by bleomycin (almost equal to the frequency of induced reciprocal translocations following exposure to 3 µg/ml bleomycin). We have earlier observed that high LET radiation, namely neutrons (1 MeV), is more efficient in inducing interstitial translocations compared with X-rays (Grigorova et al., 1998). The similar findings in neutron- and bleomycin-treated Chinese hamster cells imply that bleomycin behaves differently from low LET radiation (such as X-rays) but similarly to high LET radiation in induction of some specific types of chromosomal exchanges.
Arm-specific probes employed in this study enabled us to detect pericentric inversions which cannot be detected by whole chromosome painting. X-ray-induced chromosome intrachanges in human lymphocytes have been studied by FISH using arm-specific probes (Natarajan et al., 1996; Boei et al., 1998). It was found that the ratio of centric rings and pericentric inversions involving human chromosome 1 was ~1. In our previous study using Chinese hamster cells exposed to X-rays, the frequencies of centric rings and pericentric inversions involving chromosomes 3, 4, 8 and 9 were found to be similar (Xiao and Natarajan, 1999). In the present study, however, we observed that following exposure to 3 µg/ml bleomycin the frequencies of pericentric inversions involving chromosomes 3, 4, 8 and 9 were ~3.6, 1.9, 1.4 and 1.5 times more than the frequencies of centric rings, respectively. The higher frequency of pericentric inversions in comparison with centric rings is very likely to be another characteristic of bleomycin-induced chromosomal exchanges.
Non-random involvement of Chinese hamster chromosomes 3, 4, 8 and 9 in bleomycin-induced aberrations
The FISH technique is especially useful for rapid analysis of structural chromosome aberrations involving individual painted chromosomes. Based on the size of the chromosomes, the expected number of aberrations involving individual chromosomes can be estimated. In general, chromosomes 3 and 4 and chromosomes 8 and 9 of Chinese hamster are similar in size (Xiao and Natarajan, 1999). Therefore, a similar increase in the frequencies of aberrations involving chromosomes 3 and 4 and chromosomes 8 and 9 is expected following bleomycin treatment. In a study of X-ray-induced chromosomal aberrations in Chinese hamster cells using FISH and arm-specific probes, we found that chromosome 8 was more involved than expected, whereas chromosome 3 was less involved than expected (Xiao and Natarajan, 1999). The results obtained in this study are similar to our previous X-ray data.
The possible basis for the heterogeneity of involvement of individual chromosomes and chromosome arms in induced chromosomal aberrations has been discussed elsewhere (Xiao and Natarajan, 1998). The differential involvement of individual chromosomes studied in bleomycin-induced aberrations may be attributed to the characteristics of these chromosomes, namely chromatin condensation (most of chromosome 9 is heterochromatic) and concentration of interstitial telomeric repeat sequences (chromosome 8 is rich in telomeric repeat sequences).
In conclusion, the use of arm-specific probes to detect bleomycin-induced chromosomal aberrations in Chinese hamster cells has revealed some specific characteristics of chromosomal exchanges induced by this agent, i.e. relatively higher frequencies of interstitial translocations and pericentric inversions in comparison with those induced by X-rays, which cannot be detected by solid staining and/or whole chromosome painting.
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Acknowledgments |
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This work was supported by the European Commission Nuclear Safety Programme (contract no. CT950001 to A.T.N.).
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Notes |
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1 To whom correspondence should be addressed: Tel: +31 71 5276164; Fax: +31 71 5221615; Email: natarajan{at}rullf2.medfac.leidenuniv.nl
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References |
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Received on October 5, 1998; accepted on February 26, 1999.
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