Mutagenesis, Vol. 14, No. 5, 491-496,
September 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Analysis of chromosome loss and non-disjunction in cytokinesis-blocked lymphocytes of 24 male subjects
Laboratory of Comparative Toxicology and Ecotoxicology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, 1 Department of Genetics and Molecular Biology and 2 Center for Evolutionary Genetics,c/o Department of Genetics and Molecular Biology, Università `La Sapienza', Rome and 3 Department of Biology, Università `Roma Tre', Rome, Italy
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Abstract |
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Chromosome malsegregation in peripheral blood lymphocytes of 24 healthy male subjects was analysed by means of fluorescence in situ hybridization with centromeric probes of chromosomes 7, 11, 18 and X. On the basis of the distribution of centromeric signals in cytokinesis-blocked cells, both loss (leading to centromere-positive micronuclei) and non-disjunction (resulting in an unbalanced distribution of signals in the main nuclei) of the hybridized chromosomes in vitro were identified. In addition, the incidence of binucleated cells with two hyperploid nuclei, possibly arising from mitotic division of trisomic types, was determined. In this way, the incidence of chromosome malsegregation in vivo and in vitro could be compared in the same cell samples. The results obtained show that ageing is positively correlated with the incidence of malsegregation of chromosome X in peripheral lymphocytes of male subjects and confirm the higher susceptibility of chromosome X to malsegregation in comparison with autosomes. A positive correlation between in vitro and in vivo malsegregation rates was observed for both chromosome X and for autosomes. Finally, relatively high frequencies of multiple malsegregation events, greater than expected for independent events, were recorded for both chromosome X and for autosomes, indicating that the abnormal segregation of chromosomes may be connected to a general dysfunction of the mitotic apparatus. The correlation observed between in vitro and in vivo malsegregation frequencies and the association of both parameters with ageing suggest that analysis of chromosome malsegregation in binucleated cells is a useful tool in the study of genomic instability in human populations.
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Introduction |
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The biomonitoring of human exposure to chemical and physical genotoxins has been traditionally based on the analysis of structural chromosomal aberrations in peripheral blood lymphocytes. However, due to the acknowledged role of numerical chromosomal aberrations in carcinogenesis (Tsutsui et al., 1984
; Li et al., 1997), there is now growing concern as to the possible influence of environmental factors on genomic stability, beyond chromosome integrity. This concern has stimulated efforts at the development of biomarkers capable of detecting the induction of numerical aberrations in somatic cells of human populations at risk. Recently, the introduction of fluorescence in situ hybridization (FISH) using centromeric probes has made possible the analysis of chromosome copy number in large samples of interphase nuclei from cultured human lymphocytes (Eastmond and Pinkel, 1990) and somatic cells from exposed individuals (Rupa et al., 1995; Moore et al., 1996; Zhang et al., 1996). A significant step forward in the development of reliable methods for the analysis of genomic stability has been provided by the application of FISH methods to cytokinesis-blocked cells by means of cytochalasin B treatment. In this way, both abnormal segregation patterns of chromosomes, as well as loss of micronuclei, can be easily visualized. This approach has been successfully applied to both human lymphocytes treated in vitro (Migliore et al., 1993; Kirsch-Volders et al., 1996; Zijno et al., 1996c) and to blood lymphocytes of occupationally exposed subjects (Surrallés et al., 1997).
However, even though the above-mentioned studies suggest a potential role of FISH-based methods in the assessment of genomic stability in human populations, further work is still required for their validation. In particular, information on inter-chromosomal, tissue to tissue and inter-individual variations in the end-points analysed, as well as on potential confounding effects such as age and lifestyle factors, are still scanty. To address some of these issues, in this study in situ hybridization methods have been used for a detailed analysis of chromosome segregation in peripheral blood lymphocytes of 24 male subjects. Mitogen-stimulated lymphocytes were treated with cytochalasin B in order to obtain binucleated cells at first mitotic division. Two different centromeric probe mixes, i.e. chromosomes 7 and 11 and chromosomes X and 18, were used. Numerical alterations of these chromosomes are recurrent in human malignancies (Sandberg, 1990). On the basis of the distribution pattern of centromeric probe signals, both specific chromosome loss (i.e. centromere-positive micronuclei) and chromosomal non-disjunction (revealed by an unbalanced distribution of signals in the daughter nuclei) occurring in vitro were detected. In addition, binucleated cells with both trisomic nuclei, arising from mitotic division of parent trisomic cells, were identified and recorded, in order to provide an estimate of the frequency of specific chromosome hyperploidy before in vitro stimulation. In this way, the relative incidence of specific chromosome malsegregation in vitro and in vivo could be compared.
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Materials and methods |
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Study population
A random sample of 12 male gasoline service station attendants and 12 employees from the Istituto Superiore di Sanità (Rome), not involved in laboratory work, formed the study population. For all subjects, detailed information on smoking habit, diet and other possible confounding factors was recorded by questionnaire. The same subjects had previously been involved in a biomonitoring study on genetic effects produced by petroleum fuel exposure (Carere et al., 1998
). Since the previous study, as well as the analysis of non-disjunction, chromosome loss and hyperploidy carried out within this work, did not show any significant effect of occupational exposure, the two groups were pooled for further analyses on genomic instability.
Cell culture, chemical treatment and slide preparation
Lymphocyte cultures were established with 0.5 ml whole blood, added to 4.5 ml of RPMI 1640 medium (Gibco BRL, Paisley, UK) with 25 mM HEPES buffer and L-glutamine, supplemented with 20% heat-inactivated fetal calf serum (Hyclone, Logan, UT), 1% penicillin (5000 U/ml) and streptomycin (5000 µg/ml) solution (Flow Laboratories, UK) and 2% phytohaemagglutinin HA-15 (MUREX, Dartford, UK) and incubated at 37°C. Cytochalasin B (6 µg/ml) was added after 44 h cultivation and cells were harvested 22 h later. Lymphocytes were harvested by centrifugation (5 min at 150 g), resuspended in 0.075 M KCl, incubated for 2 min at room temperature and then gently fixed three times with methanol:acetic acid (5:1). Slides were prepared and stored at –20°C in sealed boxes until in situ hybridization.
Fluorescence in situ hybridization (FISH)
FISH with chromosome-specific probes was performed using commercial probes (Oncor, Gaithersburg, MD) for the alphoid sequences of human chromosomes 7, 11, 18 and X. Probes (10 ng/µl) were diluted in Hybrisol VI (Oncor) to a final concentration of 1 ng/µl. Two hybridization mixes were prepared: one for chromosomes 7 (biotinylated probe) and 11 (digoxigenated probe), the other for chromosomes X (biotinylated probe) and 18 (digoxigenated probe). Detection of the biotin-labelled probe was performed with 20 µg/ml fluorescein-conjugated avidin (FITC–avidin) (Vector Laboratories, Burlingame, CA) and amplification of the signal was obtained using 10 µg/ml biotinylated anti-avidin D (Vector Laboratories) and FITC–avidin. Detection of digoxigenin-labelled probes was performed using sequential application of 20 µg/ml rhodamine-conjugated mouse anti-digoxigenin (Boehringer Mannheim, Germany), Texas red rabbit anti-sheep and Texas red goat anti-rabbit antibodies. DNA was counterstained with 1 µg/ml 4,6-diamidino-2-phenylindole in antifade solution (Vectashield; Vector Laboratories).
Slide scoring
About 1000 binucleated cells/individual, hybridized with probes for chromosomes 7 and 11, were scored. In order to obtain a comparable number of analysed chromosomes for the X chromosome to those analysed for chromosome 7 and 11, which are of similar size, 2000 cells/individual, hybridized with probes for chromosomes X and 18, were scored. In this way, 10 000 chromosomes/individual, corresponding to >200 metaphases, were analysed.
Scoring criteria
After in situ hybridization with centromere-specific probes, binucleated cells with the expected number of signals were classified as normal, when all signals were evenly distributed in the two daughter nuclei, or as malsegregated in vitro, when one or more signals were found in micronuclei (chromosome loss) or when an unbalanced distribution of signals between the main nuclei was observed (non-disjunction). According to criteria previously established (Zijno et al., 1996a), cells showing malsegregation of one homologue of any of the chromosome pairs scored were classified as single malsegregation events, whereas cells showing concurrent malsegregation of different chromosomes or of both homologues of one chromosome were classified as multiple malsegregants. Binucleated cells with an abnormal number of signals in both daughter nuclei were considered putative hypodiploid and hyperdiploid, resulting from the mitotic division of pre-existing aneuploid types, arisen in vivo. Only hyperdiploid cells were considered, to avoid bias due to technical artefacts. Binucleated cells showing a double number of signals for both chromosomes were classified as polyploid. All binucleated cells with an odd number of signals were considered artefacts and rejected.
Statistical analysis
The Kolmogornov/Smirnov test showed a distribution of data different from normality for most parameters, particularly those showing low absolute values, such as autosome chromosome loss and hyperploidy. Therefore, non-parametric tests were used to analyse both the correlation between cytogenetic markers and age (Spearman rank test) and the differences in frequencies of the parameters considered (Mann–Whitney U-test).
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Results |
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The study group consisted of 24 healthy males, with a mean and median age of 45 years (range 31–62 years). Five subjects were smokers and seven former smokers. Other relevant characteristics of the study population, mainly regarding occupational exposure, are reported elsewhere (Carere et al., 1998
).
An overview of the results of the analysis of in vitro and in vivo chromosome malsegregation in the whole study population is shown in Figure 1. The figure shows the box plot of individual frequencies of non-disjunction, specific chromosome loss and hyperploidy. The comparison between the frequencies of chromosome non-disjunction (Figure 1A) and chromosome loss (Figure 1B) shows that, in the case of autosomes, non-disjunction accounted for the great majority of aberrant binucleated cells. Indeed, chromosome loss was almost negligible for autosomes. Conversely, comparable frequencies of chromosome non-disjunction and chromosome loss were found for chromosome X, indicating that chromosome X is more prone than autosomes to be lost during growth in vitro. Furthermore, chromosome X was also found to be significantly more prone than chromosomes 7 (P < 0.01) and 18 (P < 0.001) to non-disjunction in vitro. For all chromosomes studied, the incidence of unbalanced in vitro non-disjunctions was found to be higher than the frequency of balanced hyperploid cells resulting from non-disjunctional events in vivo (cf. Figure 1A and C).
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Figure 2
shows the relationship between malsegregation of chromosome X and age of the subjects. When tested by the Spearman correlation test, age was positively correlated with both chromosome non-disjunction and hyperploidy (P = 0.017 and 0.003, respectively) and close to significance for chromosome loss (P = 0.06). Figure 3 shows the correlation between non-disjunction (Figure 3A) and hyperploidy (Figure 3B) of pooled autosomes and age. Probability values close to significance were obtained for both non-disjunction and hyperploidy (P = 0.051 and 0.063, Spearman correlation test). As far as individual autosomes are concerned, non-disjunction and hyperploidy of chromosome 11 were significantly associated with age of the subjects (P = 0.042 for both end-points). On the other hand, no clear association was observed between malsegregation of chromosomes 7 and 18 and ageing (data not shown). The incidence of polyploidy was also evaluated using both chromosome mixes. The results obtained show a significant correlation in polyploidy rates determined using the two probe mixes (P = 0.009), with no significant association with the age of subjects (data not shown).
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Neither in vivo nor in vitro chromosome malsegregation frequencies showed any relationships with smoking habit. However, the small number of subjects investigated (five smokers, seven former smokers and 12 non-smokers) do not allow definitive conclusions on the role of smoking in genomic stability to be drawn.
Interestingly, a significant association was observed between individual malsegregation data in vitro (non-disjunction) and in vivo (hyperploidy), both for chromosome X (P = 0.002; Figure 4A) and for the pooled autosomes (P = 0.036; Figure 4B). Considering each autosome separately, a positive correlation between in vitro and in vivo malsegregation was observed for chromosome 18 (P = 0.021) and a tendency toward significance for chromosomes 7 and 11 (P = 0.092 and 0.158, respectively).
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An important advantage offered by multicolour FISH is the possibility of identifying malsegregation events involving more than one chromosome. The results obtained in this study with simultaneous analysis of two chromosome pairs are summarized in Table I
. `Single events' indicates the number of binucleated cells with a single homologue malsegregated. `Multiple events', according to the criteria detailed in Materials and methods, indicates the number of cells with simultaneous malsegregation of both homologues (e.g. Ch 18–18, Ch 11–11, etc.) or with malsegregation of both hybridized chromosomes (i.e. Ch X–18 and Ch 7–11). Expected frequencies of multiple malsegregation events were calculated according to the hypothesis of independence, multiplying the overall frequencies of each of the malsegregation events involved. This value is given by the sum of the observed malsegregation events involving one homologue, plus twice the number of events involving both homologues. As shown in Table I, high frequencies of malsegregation events involving two chromosomes were recorded. The frequencies of multiple events detected by both the chromosomes X and 18 mix and the chromosomes 7 and 11 mix were 62- and 32-fold higher than expected on the hypothesis of random, independent events.
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Discussion |
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In this study the inter-individual variation in potential biomarkers for genomic instability was investigated in cytokinesis-blocked cells hybridized with centromeric DNA probes. Chromosome-specific probes were used to assess the susceptibility to malsegregation of four chromosomes in peripheral lymphocytes from 24 male subjects. The parallel analysis of chromosome non-disjunction (resulting from in vitro malsegregation) and hyperploidy (secondary to in vivo malsegregation events) in binucleated cells provided the opportunity to compare chromosome malsegregation at mitosis in vivo and during lymphocyte growth in culture.
The results obtained confirm the greater susceptibility of chromosome X to malsegregation with respect to autosomes. Chromosome X was particularly prone to malsegregate in vitro, where it accounted for most observed malsegregation events, despite its haploid status in male cells. Chromosome X displayed similar rates of non-disjunction and loss, at variance with the autosomes, which showed negligible rates of loss compared with non-disjunction. The incidence of hyperploidy of chromosome X in peripheral lymphocytes was positively correlated with the age of the subjects (age range 31–62 years), confirming the results of previous studies which showed increased frequencies of chromosome loss and non-disjunction of chromosomes X and Y in lymphocytes of aged males (Nath et al., 1995; Zijno et al., 1996b; Catalán et al., 1998). The evidence provided by these studies in male subjects indicate that the relatively high and age-related propensity of chromosome X to malsegregate is not related to its inactivation status. Similar conclusions were drawn from studies in females (Surrallés et al., 1996b; Zijno et al., 1996a), which failed to demonstrate preferential malsegregation of one X homologue. In this study the incidence of hyperploidy of the autosomic chromosomes showed a possible association with age of the subjects, while polyploidy rates were unrelated to age. Even though the molecular mechanism underlying age-related loss of fidelity in chromosome segregation has not been elucidated, some clues come from parallel analysis of chromosome malsegregation in vitro. The results obtained show that ageing was also correlated with in vitro malsegregation rates of chromosome X and autosomes, indicating that the age-related loss of fidelity in chromosome segregation is an intrinsic cellular trait, rather than a consequence of altered environmental conditions (e.g. decreased defences against oxidative stress, etc) in the living organism.
A comparison of the frequencies of non-disjunctionals (i.e. in vitro malsegregants) and hyperploids (i.e. pre-existing malsegregants) in the same subject highlighted higher malsegregation rates for all chromosomes in vitro than in vivo. The effect of cytochalasin B on chromosome loss and non-disjunction is still a matter of debate. It has been suggested that cytochalasin B interferes with anaphase B, reducing the distance between spindle poles and causing engulfment of laggards by the nearest daughter nucleus (Norppa et al., 1993; Surrallés et al., 1996a; Minissi et al., 1999), thereby increasing the frequency of non-disjunctional cells in half of the cases. However, a short pole to pole distance may also result in polyploid nuclei and this may reduce the observed frequencies of non-disjunction in binucleated cells (Cimini et al., l999). We believe that more stringent control of the survival of aneuploid cells in vivo, especially with respect to heavily unbalanced cells, is a more plausible explanation for the observed differences. In any case, it should be pointed out that the significant correlation observed between in vivo and in vitro overall malsegregation rates among the study subjects indicates that a basically similar mechanism(s) controls the fidelity of chromosome segregation in vivo and under culture conditions.
The analysis of multiple events shows that ~9% of cells showing malsegregation of chromosome X also showed malsegregation of chromosome 18. Considering the number of autosome pairs in the cell, it follows that the great majority of cells showing malsegregation of chromosome X should show malsegregation of another chromosome(s) as well. This suggests the involvement of a mechanism(s) disturbing the fidelity of the whole mitotic process, which in most cases affects multiple chromosomes. Simultaneous analysis of chromosomes 7 and 11 also showed a relatively high incidence of multiple malsegregation events, far above that expected on the hypothesis of independent events, thus confirming the hypothesis that abnormal chromosomal segregation is connected to a general misfunctioning of the mitotic apparatus.
A comparison of different malsegregated types shows that mitotic non-disjunction contributes to a larger extent, compared with chromosome loss, to in vitro chromosome malsegregation in human lymphocytes. This finding is in agreement with the results of studies on chemically and physically induced aneuploidy, which demonstrated that non-disjunction occurs at lower concentrations than chromosome loss (Kirsch-Volders et al., 1996; Marshall et al., 1996; Zijno et al., 1996c; Elhajouji et al., 1997; Sgura et al., 1997). As a consequence, the analysis of chromosome misdistribution in binucleated cells, using FISH methods and chromosome-specific centromeric probes, should be a more sensitive approach, compared with the scoring of centromere-positive micronuclei, for the assessment of the effects of environmental agents on genomic stability. As to the choice of marker chromosome(s), the high susceptibility of chromosome X to both spontaneous and chemically induced malsegregation (Marshall et al., 1996; Xi et al., 1997) suggests that, in the first instance, this chromosome represents a convenient marker for studies on human populations exposed to environmental agents. However, since it is recognized that aneugenic chemicals may differentially affect specific chromosomes in relation to their specific mechanism of action (Xi et al., 1997), other chromosomes should be simultaneously analysed to increase the sensitivity and reliability of the analysis. Multicolour FISH with chromosome-specific probes may represent a useful tool to this end, allowing the evaluation of the effects of environmental agents on genomic stability in human populations with accuracy and reliability.
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Acknowledgments |
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D.C. is a doctoral fellow of the Dipartimento di Genetica e Biologia Molecolare, Università `La Sapienza' Rome. This study was partially funded by the EU (contract no. EV5V-CT92-0221) and by the Italian Ministry of the Environment (project PR 22-IS).
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Notes |
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4 To whom correspondence should be addressed. Tel: +39 06 49902762; Fax: +39 06 49387139; Email: acarere{at}iss.it
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References |
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Received on February 2, 1999; accepted on June 7, 1999.
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