Mutagenesis, Vol. 15, No. 6, 459-467,
November 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Chromosome painting reveals specific patterns of chromosome occurrence in mitomycin C- and diethylstilboestrol-induced micronuclei
Abt. Humanbiologie und Humangenetik der Universität, Postfach 3049, D-67653 Kaiserslautern, Germany
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Abstract |
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Cultures of human blood lymphocytes from three subjects were incubated with the clastogen mitomycin C (MMC, 500 ng/ml) and the aneugen diethylstilboestrol (DES, 80 µM) 23 h before harvesting, to induce formation of micronuclei (MN) and numerical and structural alterations in metaphase chromosomes. We used fluorescence in situ hybridization (FISH) with painting probes for all human chromosomes to determine which chromosomes had contributed material to the induced MN. MMC treatment induced an ~18-fold increase in MN and led to a significant increase in hypodiploidy and structural chromosome aberrations in metaphase preparations. Undercondensation of pericentromeric heterochromatin of chromosomes 9 and 1 occurred in 20–75% of metaphases and FISH disclosed an abundance of material from these chromosomes in induced MN (62–69% from chromosome 9 and 7–12% from chromosome 1). DES treatment of lymphocytes induced a seven-fold increase in MN frequency and four-fold increase in the frequency of numerical aberrations; structural aberrations were not significantly increased. FISH analysis showed that material from all chromosomes was present in DES-induced MN, with material from chromosome 1 present in 16% of MN and material from each other chromosomes being present in 2–10% of MN. Material from chromosomes 14, 19 and 21 was significantly more frequent material from chromosome Y significantly less frequent in DES-treated cells than in controls. The findings of the MMC studies indicate that the heterochromatin block of chromosome 9 is a specific target for MMC-induced undercondensation, which induces a preferential occurrence of chromosome 9 material in MN. DES, in contrast, does not trigger heterochromatin decondensation and fails to induce such a significant appearance of material of particular chromosomes in MN.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The micronucleus (MN) test is a well established assay for mutagenicity testing, since clastogens (chromosome-breaking agents) and aneugens (spindle poisons) have been shown to increase the number of MN in post-mitotic cells (for review see Heddle et al., 1991; Kirsch-Volders et al., 1997). Aneugenic and clastogenic effects can be distinguished to some extent by staining the centromeric regions of chromosomes found in MN: whole chromosomes, which are preferentially included in MN after treatment with aneugens, contain centromeres. Acentric fragments, which result from chromosome breakage, will generally give rise to centromere-negative MN. Centromeres in MN can be revealed by immunofluorescence techniques with CREST serum by staining kinetochore proteins (Degrassi and Tanzarella, 1988
; Eastmond and Tucker, 1989) or by FISH with pan-centromeric DNA probes (Becker et al., 1990, Miller et al., 1992). The drugs that have been studied in most detail for their mechanism of MN induction are the clastogen mitomycin C (MMC) (e.g. Miller and Adler, 1990; Miller et al., 1991; Rudd et al., 1991; Surrallés et al., 1995) and the aneugen diethylstilboestrol (DES) (e.g. Eastmond and Tucker, 1989; Migliore et al., 1996).
Many studies have addressed the mechanisms of MN induction (see above), but only a few investigators have examined the chromosomal content of MN. The latter studies have shown that some mutagens induce preferential occurrence of particular chromosomes in MN. Chromosomes 1, 9, 15, 16 and Y were found in elevated frequencies in 5-azacytidine-induced MN (Guttenbach and Schmid, 1994; Fauth et al., 1998), chromosome 9 in idoxuridine-induced MN (Tommerup, 1984; Fauth and Zankl, 1999), chromosome 7 in colcemid-induced MN (Wuttke et al., 1997) and acrocentric chromosomes in vanadium- and colcemid-induced MN (Migliore et al., 1995; Caria et al., 1996).
Using FISH analysis with whole chromosome painting probes (WCPs) for all human chromosomes (Fauth et al., 1998), we investigated which chromosomes had contributed material to induced MN in MMC- and DES-treated short-term human lymphocyte cultures. WCPs target the euchromatic parts of a chromosome and thereby disclose both whole chromosomes and acentric fragments in MN. However, they fail to distinguish between an entire chromosome or material from a large chromosomal fragment(s) in a particular MN.
Giemsa-stained metaphases were examined for numerical and structural chromosome alterations to obtain insight into the chromosome-specific effects of the mutagens investigated. For instance, MMC is reported to induce preferentially breakage and undercondensation in the heterochromatin blocks of chromosomes 1q12, 9q12 and 16q12 (Cohen and Shaw, 1964; Nowell, 1964; Savage and Reddy, 1987). We were particularly interested in a possible correlation of MMC-induced undercondensation of characteristic regions of metaphase chromosomes and the frequency with which material from particular chromosomes was found in MN. We also wanted to compare the effects of MMC and DES treatment.
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Materials and methods |
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Probands and preparation cell cultures
Peripheral blood (10 ml) was obtained from three donors (two females and one male, hereafter referred to as `fem1', `fem2' and `male', respectively). All subjects had normal karyotypes (not shown) and were 22–35 years of age. Cultures denoted fem1 in this study and those denoted fem1 in a previous study (Fauth et al., 1998
) were obtained from the same individual. Several parallel lymphocyte cultures were set up in each experiment. Repeated experiments were performed with blood from subject fem1 (for details see Table I).
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For each culture, 0.8 ml peripheral blood was added to 8 ml RPMI 1640 medium (supplemented with 300 µg/ml glutamine; Bio Whittaker) containing 17% fetal calf serum (Life Technologies), 1% penicillin–streptomycin (Bio Whittaker) and 0.2 ml phytohemagglutinin (22 µg/ml; Life Technologies). Duplicate cultures were set up for chromosome painting of MN (MN-FISH). After harvesting (see below), cell suspensions were pooled, to give the large number of cells required for MN-FISH. Therefore, duplicate cultures could not be comparatively analysed.
Drug treatment
Twenty-three hours before harvest, subcultures were supplemented with either (i) MMC (Sigma M-0503; final concentration 500 ng/ml culture; dissolved in 0.9% NaCl solution, giving a final NaCl concentration of 0.02% in culture) or (ii) DES [Sigma D-4628; final concentration 80 µM; dissolved in dimethylsulfoxide (DMSO), resulting in a final concentration of 1% DMSO in the culture].
One DES control culture was supplemented with 1% DMSO, since DES was set up in this solvent (Table I
). In some experiments, control cultures with and without DMSO were used to study the effects of this solvent. Since MMC was dissolved in physiological (0.9%) NaCl, solvent controls were not set up for MMC control cultures.
Cultivation and harvesting
Lymphocyte cultures were incubated for 71 h at 37°C. Colcemid solution (final concentration 0.22 µg/ml) was added to all cultures 2 h before harvesting. Finally, cultures were hypotonically treated (for 20 min with 0.0375 M KCl at 37°C) and fixed three times in methanol–acetic acid (3:1). For analysis of metaphase alterations and MN frequencies in the same preparations, these slides were stained with Giemsa according to standard methods. Because this protocol also allows one to control the specificity of the FISH signals in MN by examination of the hybridization efficiency of adjacent metaphases (see below), we accepted the minor drawback that this method does not identify metaphases from the second cell cycle, as has been recommended for aneuploidy detection (Parry et al., 1995).
Scoring of MN frequencies
MN frequencies were scored in ~5000 cells of Giemsa-stained slides prepared from control and the respective MMC-treated cultures of fem1, fem2 and male cultures. About 3000 cells of DES-treated cultures and the respective controls of fem1 and male were examined in the same way. Repeated experiments were performed to evaluate spontaneous MN frequencies in subject fem1.
Cells were regarded as micronucleated when fulfilling the criteria of Countryman and Heddle (1976) with the following modification: the distance between a MN and the corresponding main nucleus was requested to be less than or equal to the diameter of the main nucleus. We applied this stringent distance restriction to cope with the complication that methanol:acetic acid fixation, which removes nearly all the cytoplasm, leaves it difficult to assign a particular MN to a more distant main nucleus. Despite this more rigid criterion, it is feasible that MN could have become displaced across the slide and end up near the nucleus of another cell. However, since there is no way to score the slides with cytoplasm, and because the unit of analysis was the MN and not the cell, we regarded this drawback as acceptable.
Scoring of metaphase alterations
At least 50 Giemsa-stained metaphases from MMC-treated cultures as well as corresponding control cultures (CM) of fem1, fem2 and male and from DES-treated and corresponding control cultures (CD) of fem1 and male were analysed for the presence of numerical aberrations (hypo- and hyperdiploidy).
Structural changes (breaks, gaps, acentric fragments, exchanges and undercondensed regions) were analysed also in at least 50 Giemsa-stained metaphases from matching controls and MMC-treated cultures of male, fem1 and fem2 as well as DES-treated cultures of fem1 and the male subject and respective controls. Chromosomes were classified as `undercondensed' when the pericentromeric heterochromatin (qh) of chromosomes appeared extended for more than a chromatid width (Schmid et al., 1984).
DNA probes and labelling
WCP plasmid library DNA probes for all human chromosomes (Collins et al., 1991) were kindly provided by J.W.Gray (University of California, San Francisco). Plasmid DNA was obtained by midi-scale preparation according to Kaul and Scherthan (1990). Probe DNA was labelled with biotin-16-dUTP and digoxigenin-11-dUTP using Biotin- and Dig-Nick Translation Mix according to the manufacturer's protocol (Roche Biochemicals).
FISH
FISH on MN was performed according to Fauth et al. (1998). Briefly, differentially labelled WCP DNA probes were applied in pairwise combinations (i.e. dual-colour FISH with two probes on the same slide). Forty microlitres of hybridization mixture [containing 1 µg of biotinylated and 1 µg of digoxigenated WCP DNA probe, 2 µg herring sperm DNA (Roche Biochemicals), 20 µg human C0t-1 DNA (Life Technologies), 50% formamide, 10% dextran sulfate, and 2x SSC were applied per slide and sealed under a 22x60 mm coverslip with rubber cement. Preparations were denatured on a hot plate at 78°C for 200 s.
For in situ hybridization, slides were incubated for 48 h at 37°C. After removal of rubber cement and coverslip, slides were washed three times for 5 min in 0.05x SSC at 42°C (Scherthan and Cremer, 1994). Biotinylated hybrid molecules were detected with fluoresceinated avidin (Sigma) and digoxigenin-labelled hybrid molecules with rhodamine anti-digoxigenin Fab fragments (Roche Biochemicals) (for details see Scherthan and Cremer, 1994). Finally, the slides were mounted in Vectashield (Vector) containing 1 µg/ml 4',6-diamidino-2-phenylindole (DAPI). This DAPI staining protocol was also applied to scrutinize slides of MMC- and DES-treated cultures of fem1 for MN with DAPI-positive heterochromatin blocks (see below).
Microscopic evaluation
At least 50 MN with associated interphase nuclei were scored for FISH signals. To avoid misinterpretations, only preparations with strong specific signals in both MN and neighbouring nuclei as well as metaphases were investigated. The drug-treated cultures of subject fem1 were analysed with all human WCPs. The occurrence of material from chromosomes 1, 6, 9, 15, 16 and X was also determined in MMC-induced MN of subject fem2. The frequency of Y-chromosome material was determined in drug-treated and control cultures of the male proband.
Preparations were examined with a Leitz Orthoplan research microscope equipped with a Ploem Opak fluorescence epi-illumination system, filter blocks A and I2 for excitation of green and red fluorescence and a green/red double band pass filter for simultaneous excitation of both emissions (Chroma).
Analysis of DAPI-bright heterochromatin of MN
As a result of its staining preference for AT-rich DNA sequences, DAPI stains particularly strongly at the heterochromatin blocks of chromosomes 1q12, 9q12, 16q12 and Yq12 (see Schweizer, 1981; Francke, 1994). These regions are seen as bright blue clusters/spots in interphase nuclei, particularly after the denaturation step of the FISH protocol. These `DAPI-bright' spots can be readily identified in MN and indicate the presence of pericentromeric heterochromatin material from chromosomes 1, 9, 16 or Y (Guttenbach et al., 1994). Besides the DAPI-bright heterochromatin blocks, weak blue fluorescence indicates the presence of euchromatic chromosome arm material in MN. In the present investigation we scored the occurrence of DAPI-bright heterochromatin blocks in MN, which are referred to as `DAPI-bright MN'.
Statistical analysis
Statistical evaluations were performed by t-test (MN frequencies) or 
2-test (metaphase alterations, MN-FISH studies).
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Results |
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DMSO does not affect the frequency of spontaneous MN formation
Comparison of the spontaneous MN frequencies in control cultures without and with 1% DMSO from fem1 (0.2 ± 0% and 0.1 ± 0.08%, respectively) and the male subject (0.03 ± 0.05% and 0.07 ± 0.5%, respectively) showed no significant difference between the cultures, indicating that DMSO does not increase the frequency of MN formation.
MN frequency after treatment of human lymphocytes with MMC or DES
Four lymphocyte cultures from three subjects (two from fem1 and one each from fem2 and the male) treated with MMC (500 ng/ml) displayed MN frequencies of 2.5–5.2%. This is 18-fold higher than the frequency of MN in control cultures (0.2%), a highly significant increase (P < 0.001) (Table II).
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DES-treated lymphocyte cultures (n = 6, five from fem1 and one from the male subject) displayed MN frequencies of 1.3–3.6%, which represents a significant (P < 0.001 or P < 0.01) increase over control values (0.2–0.4%; Table II
). Statistically significant differences in spontaneous MN frequencies within and between the different subjects were not observed. In contrast, MN frequencies obtained by MMC and DES treatment showed significant differences within individuals (up to P < 0.05) and between individuals (up to P < 0.001). The latter observations may reflect individual sensitivities of the lymphocytes of the different subjects to the applied drugs (see Wuttke et al., 1993).
Metaphase analysis after incubation with MMC or DES
Metaphases of untreated lymphocytes of fem1, fem2 and the male subject showed few numerical and structural aberrations. Few metaphases (0–8%) displayed elongations and weakly stained regions in the heterochromatin blocks of different chromosomes, which are diagnostic of defects in heterochromatin condensation (Tables III and IV).
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MMC treatment led to a 4- to 5.7-fold increase in the frequency of numerical aberrations over control (Table III
). The vast majority of numerical aberrations were hypodiploidies, while 
2% of metaphases in the MMC-treated cultures were hyperdiploid (Table III).
MMC treatment induced undercondensations in 64–88% of metaphases of all three subjects, which represent highly significant (P < 0.001) increases as compared with control (Table III). The undercondensations primarily concerned the pericentromeric heterochromatin of chromosomes 9q12, 1q12 and 16q12 (Table III). Chromosome 9 was most heavily affected: both homologues frequently displayed undercondensed heterochromatin blocks (Table III). Other structural aberrations, absent in the controls, were found in 34–46% of MMC-treated metaphases (Table III).
DES treatment of lymphocytes from the fem1 and male subjects increased the total frequency of numerical metaphase aberrations 2.75- to 5.5-fold over control (P < 0.001), with significant differences in both hypodiploidy and hyperdiploidy (Table IV).
DES-treated cultures and corresponding controls from the fem1 and male subjects displayed similar low frequencies of structural metaphase chromosome alterations (Table IV).
Frequency of occurrence of chromosome material
MMC-induced MN. Chromosome painting of MN revealed that MMC-induced MN in cultures from fem1 predominantly contained material of chromosomes 1 (12%) and 9 (62%). These frequencies are two- and six-fold higher than control values, respectively (Table V), with the increase in the occurrence of chromosome 9 material being highly significant (P < 0.001). Material from other chromosomes was found in 1–6% of MN, while chromosomes 2, 3, 8, 10–12, 14, 17 and 18 were not detected in MN (Table V). A significant (P < 0.05) decrease was noted for the occurrence of chromosome 3 and X material in MMC-induced MN (Table V; control values of fem1 were taken from Fauth et al., 1998). Finally, we detected Y chromosome material in 0.9% of male MMC-induced MN, which represents a significant (P < 0.001) decrease as compared with control (11%; Table V).
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MMC treatment of lymphocytes of fem2 also induced MN. Again, the increase in occurrence of chromosome 9 material in MMC-induced MN (69% compared with 14%; Table VI
) was significant (P < 0.001). We also determined the occurrence of material from chromosomes 1, 6, 15, 16, and X in MMC-induced MN. Chromosomes 1, 15 and 16 contain heterochromatin blocks, and the other two chromosomes served as controls without large heterochromatin blocks. As compared with the control, no significant alterations in the occurrence of material from these five chromosomes were noted in MMC-induced MN of fem2 (Table VI).
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DES-induced MN. MN-FISH analysis of DES-induced MN of fem1 revealed that material from each human chromosome was present in the MN studied (Table V
). Most frequently, material of chromosome 1 (16%) was observed. Material from chromosomes 14, 19 and 21 was also found significantly (P < 0.05) more often than in controls (Table V; control values taken from Fauth et al., 1998). Seven per cent of the DES-induced MN of the male subject contained Y chromosome material, significantly (P < 0.05) less than the control (Table V).
Heterochromatin content
MMC-induced MN. Sixty-six per cent of MMC-induced MN (n = 353) of fem1 were positive for DAPI-bright heterochromatin clusters (Table VII), compared with only 12% of control (value taken from Fauth et al., 1998) (P < 0.001). Eighty-five per cent of the chromosome 9 FISH signal-containing MMC-induced MN showed a distinct DAPI-bright spot (Table VII), again significantly (P < 0.001) more than in control. Fifty per cent of MMC-induced MN without a chromosome 9 WCP signal displayed DAPI-bright heterochromatin signals (Table VII).
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DES-induced MN. A DAPI-bright signal was found in 15% of DES-induced MN of fem1 (Table VII
), which is similar to the frequency of DAPI-bright heterochromatin signals observed in spontaneously formed MN from this subject (12%; see Fauth et al., 1998). |
Discussion |
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In the present study, we determined the occurrence of euchromatic human chromosome material and DAPI-bright heterochromatin in MN induced by 500 ng/ml MMC or 80 µM DES. We performed the MN test in standard lymphocyte preparations with cytoplasm removed by conventional acetic acid–methanol fixation, since our control experiments had revealed that remnants of cytoplasm impair penetration of the FISH probes and may cause non-specific signals (unpublished data). To compensate for the problems in assigning a MN to its main nucleus that are created by loss of nearly all cytoplasm, we applied a more stringent definition for MN identification than those suggested by Countryman and Heddle (1976). We considered MN only when they occurred within a distance of not more than the diameter from the main nucleus. Although one might expect that this more stringent criterion would lead to an underestimation of MN frequencies, we found that our drug-induced frequencies were not lower than previous MN frequencies obtained following the criteria of Countryman and Heddle (1976) in MMC- and DES-treated cultures (Surrallés et al., 1995
; Scarfi et al., 1996; Eastmond and Tucker, 1989). Spontaneous MN frequencies of lymphocyte cultures of our three subjects were in the range 0.2–0.4%, which is in good agreement with previous investigations (Fenech and Morley, 1984; Högstedt, 1984; Ghosh et al., 1990; Ganguly, 1993; da Cruz et al., 1994; Guttenbach et al., 1994; Forni et al., 1996; da Silva Augusto et al., 1997).
Some general considerations
Although the number of MN analysed here (50) may, in statistical terms, appear low, it has to be taken into consideration that investigation of the occurrence of all the human chromosomes by two-colour FISH requires the assessment of 600 well-hybridized MN. Furthermore, the metaphase analyses performed were primarily done in support of the MN investigation to reveal drug effects on metaphase chromosome structure. We are aware that harvesting all the cells at once is not ideal, as aneuploidy can only be detected reliably in the second metaphase after treatment whereas structural chromosome aberrations should be analysed in the first metaphase after treatment. Future analysis is required to address these points.
Numerical chromosome aberrations caused by MMC
MMC treatment significantly increased the occurrence of hypodiploidy in the lymphocytes of the subjects studied here. This is consistent with previous reports which showed that MMC has the potential to elicit aneugenic effects: Rudd et al. (1991) detected a four-fold increase in kinetochore-positive MN after MMC treatment of human fibroblasts. Similar findings were reported by Thomson and Perry (1988) in human lymphocytes. The increase in kinetochore-positive MN may relate to the preferential binding of MMC to heterochromatin and induction of kinetochore detachment (Brinkley et al., 1985). Since we did not see an increase in hyperdiploid metaphases after MMC treatment, it is likely that impaired kinetochore function may have induced MN formation. The MMC-induced loss of kinetochore function might be responsible for creation of MN that fail to display kinetochore immunofluorescence despite containing whole chromosomes (Miller and Adler, 1990). Therefore, the aneugenic potential of MMC may have been underestimated. Aneugenic effects of MMC have also been confirmed in polychromatic erythrocytes of bone marrow of the mouse (Schneider et al., 1995). Furthermore, some potent clastogens have also been shown to elicit weak but significant aneugenic effects (Tucker and Eastmond, 1990). Additionally, it has to be considered that technical artefacts could have led to hyperdiploid metaphases (see below).
Structural chromosome aberrations caused by MMC
Structural analysis of metaphase chromosomes from MMC-incubated blood lymphocytes revealed an increased frequency of undercondensation preferentially of the pericentromeric heterochromatin block at 9q12 and to a much lesser extent at 1q12 and 16q12. This corresponds well with earlier reports, which showed that MMC induces undercondensation and breakage in the pericentromeric heterochromatin blocks of chromosomes 1, 9 and 16 (Cohen and Shaw, 1964; German, 1964; Nowell, 1964; Cohen, 1969; Hoehn and Martin, 1972; Morad et al., 1973; Brogger, 1974a,b; Rupa et al., 1997).
The specific decondensation of 9q12 by MMC treatment corresponds well with a general sensitivity of this region to drugs which induce undercondensation and breakage like melphalan (Mamuris et al., 1991), idoxuridine (Tommerup, 1984; Ott et al., 1998; Fauth and Zankl, 1999) and 5-azacytidine (reviewed in Haaf, 1995). Since ionizing radiation induces translocations that often involve breakage of the 9q12 region (Holmberg, 1992), the heterochromatin block of this chromosome may be particularly sensitive to mutagenic impact. This feature of the 9qh region contrasts with that of other heterochromatin-containing chromosomes and could relate to the unique DNA composition of 9qh which has been shown to contain a mixture of various tandemly repeated sequences such as 
-, ß- and classical satellite DNAs I–IV (Gosden et al., 1975; Waye and Willard, 1989; Fernandez et al., 1994a,b; Lee et al., 1997). This variation gives rise to a multitude of polymorphic variants, probably caused by unequal crossing over (Metaxotou et al., 1978; Kurnit, 1979; Park et al., 1998). 5-Azacytidine treatment has revealed that -, ß- and classical human satellite DNAs are differentially susceptible to induced undercondensation (Fernandez et al., 1994a,b). However, little is known about clinical consequences of chromosome 9qh polymorphisms in man, although it has been supposed that alterations of the region 9qh, particularly inversions, might be associated with spontaneous abortions and ovarian cancer (for review see Hasegawa et al., 1995).
Frequency of human chromosome material in MMC-induced MN
The application of chromosome painting with all human WCPs to MMC-induced MN disclosed the complete chromosomal spectrum in these MN. However, the WCP applied here did not allow us to differentiate between whole chromosomes and fragments thereof. The application of simultaneous FISH with WCPs and primed in situ labelling (PRINS) with centromere probes, as described by Hindkjaer et al. (1995), would have been helpful to circumvent this problem. However, the former approach proved to be impractical for MN-FISH (unpublished observations). Alternatively, dual-colour FISH of WCPs and chromosome-specific centromeric probes or WCPs combined with a general centromeric probe could be applied. Unfortunately, combination of the pan-centromeric probe p82h (Becker et al., 1990) with WCPs was unsuccessful in our hands.
FISH analysis of MMC-induced MN from the two female donors revealed the preferential occurrence of chromosome 9 material in MN, which strongly correlated with preferential undercondensation of chromosome 9qh in metaphases of these subjects. Chromosome 1 material, in contrast, was found to be two-fold enriched in MMC-induced MN of fem1, but not in those of fem2. This discrepancy may relate to the higher levels of 1qh undercondensation in MMC-induced metaphases of fem1 (Table III). These data indicate that the frequency of heterochromatin undercondensation at metaphase correlates positively with the occurrence of chromosome material in MN but this effect seems to be threshold dependent. Perhaps the number of metaphases displaying undercondensation of a particular chromosome has to be reach a certain level before its increased occurrence becomes apparent in corresponding MN. A similar correlation has been noted in lymphocytes treated with 5-azacytidine (Guttenbach and Schmid, 1994; Fauth et al., 1998) and idoxuridine (Tommerup, 1984; Fauth and Zankl, 1999).
Heterochromatin content of MMC-induced MN
When we monitored the occurrence of heterochromatin material in MN by staining with DAPI (Guttenbach et al., 1994; Fauth et al., 1998), it was found that MMC treatment induced formation of many MN with DAPI-bright signals, which indicates an increased loss of heterochromatin-rich chromosomes or material thereof. Eighty-five per cent of MN showing chromosome 9 FISH signals contained a DAPI-bright heterochromatin signal, which makes it likely that chromosome 9 heterochromatin is also enriched in MN. However, further FISH studies with centromeric probes are required to elucidate this point. Our findings indicate that either the complete chromosome 9 was lost and occurred in a MN or that a break within or near the heterochromatic block led to its preferential exclusion from the main nucleus. The strong clastogenic effect seen in metaphases after MMC treatment would favour the second possibility. In this context it may be of interest that DNA damage in heterochromatin may be more persistent than in euchromatin, since DNA damage in non-transcribed DNA is repaired less rapidly than in transcribed DNA (for review see Bohr and Wassermann, 1988).
Preferential occurrence of DAPI-positive MN may also be triggered by drugs like 5-azacytidine and idoxuridine, which both induce heterochromatin undercondensation in metaphase chromosomes of human lymphocytes (Guttenbach and Schmid, 1994; Fauth et al., 1998; Fauth and Zankl, 1999).
DES induces elevated MN frequencies and numerical chromosome aberrations
In agreement with previous studies (Danford and Parry, 1982; Schmuck et al., 1988; Schiffmann and de Boni, 1991; Stopper et al., 1994; Migliore et al., 1996; Schuler et al., 1998), we found that treatment of peripheral lymphocytes with DES led to a significant increase in MN frequency. DES has been shown to induce numerical aberrations (e.g. Schiffmann and de Boni, 1991; de Sario et al., 1990) and is considered to act as spindle poison, inhibiting the polymerization of tubulin into microtubules (Parry et al., 1982; Hennig et al., 1988; Metzler and Pfeiffer, 1995). In the present investigation we noted that DES induced a dramatic increase in numerical aberrations, which particularly led to elevated frequencies of hyperdiploid metaphases. This contrasts with MMC, which induced solely hypodiploid metaphases. Although our observations would fit with general mis-segregation caused by DES and the action of particularly MMC at kinetochores (leading to anaphase laggards), the results have to be taken with caution, since hypodiploidy in metaphase spreads (which was particularly evident in DES-treated metaphases of the male subject) could also be the consequence of hypotonic treatment (Danford, 1984) or other technical variables (Fitzgerald and McEwan, 1977; Brown et al., 1983). Hence the observed hypodiploidy in MMC- and DES-treated metaphases could have been due, at least in part, to artificial chromosome loss. Since anaphase lagging will induce hypodiploid daughter nuclei, Ford et al. (1988) suggested that lagging is a major mechanism of chromosome loss in peripheral lymphocyte cultures in vitro. Whole chromosome loss during the anaphase transition induced by DES fits well with the observation that this drug does not induce structural chromosome aberrations (Abe and Sasaki, 1977; Bishun et al., 1977; Ishidate and Odashima, 1977; Dean, 1981; this investigation).
Frequency of human chromosome material in DES-induced MN
Our FISH study in DES-induced MN showed that material from each chromosome occurred in 2–10% of MN, except chromosome 1 material, which was found in 16% of MN. This result suggests that the occurrence of chromosomes in DES-induced MN is more random than that in clastogen–induced MN. The results obtained with DES are reminiscent of the chromosomal spectrum observed in MN induced by ionizing radiation in human lymphocytes (Fimognari et al., 1997). Although the mode of DES action is quite different from that of radiation, it might be that larger chromosomes are more likely to be lost from a DES-damaged spindle apparatus. However, our metaphase analysis revealed a preferential loss of small chromosomes from hypodiploid metaphases and a gain of A- and C-group chromosomes in hyperdiploidies (unpublished observations). Spindle disturbances may cause loss of more than one chromosome from metaphases (Hartley-Asp et al., 1985; Xiao et al., 1996) which increases the possibility that aneugen-induced MN may contain several chromosomes.
We never observed two WCP signals from two different chromosomes in a single MN. Since we used dual-colour FISH (i.e. two different probes at once) we can make no exact statement about the frequency of MN containing material from more than one chromosome. This drawback could be circumvented by multicolour in situ techniques like M-FISH or Sky (Schröck et al., 1996; Speicher et al., 1996). However, if there was one (and only one) chromosome per MN, one should theoretically get a value of 100% if all the occurrence frequencies obtained for all human chromosomes in a particular experiment were summed up. Any drug that induced MN containing material from more than one chromosome should lead to values of >100% when the percentages of the occurrence frequencies of all chromosomes in the MN are summed. We found that DES-induced MN showed a sum of all occurrence frequencies of 150%, indicating that MN induced by this aneugen on average contained material of 1.5 chromosomes, which is consistent with the proposed aneugenic effect of DES. MMC-induced MN, in contrast, contained material equal to one chromosome per MN (100%).
Preferential occurrence of DAPI-bright spots in DES-induced MN was not observed, suggesting that DES acted more randomly on chromosome loss. These results support our MN-FISH data and those of Schuler et al. (1998), which show a more random MN exclusion spectrum induced by spindle poisons.
Conclusions
MN-FISH has the potential to reveal the occurrence spectrum of the complete human chromosome complement in spontaneous and induced MN and to identify potential chromosomal targets of mutagenic substances like MMC and DES. Future investigation will have to show whether we can identify further chromosomal targets or target regions of other drugs.
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Acknowledgments |
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We would like to thank an unidentified reviewer for his comments on an earlier draft of this paper.
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Notes |
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1 To whom correspondence should be addressed. E-mail: efauth{at}rhrk.uni-kl.de
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References |
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Received on October 11, 1999; accepted on August 15, 2000.
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