Mutagenesis vol.14 no.5 pp.491–496, 1999
Analysis of chromosome loss and non-disjunction in cytokinesisblocked lymphocytes of 24 male subjects
A.Carere4, A.Antoccia1, D.Cimini2, R.Crebelli,
F.Degrassi2, P.Leopardi, F.Marcon, A.Sgura3,
C.Tanzarella3 and A.Zijno
Laboratory of Comparative Toxicology and Ecotoxicology, Istituto Superiore
di Sanità, Viale Regina Elena 299, 00161 Rome, 1Department of Genetics
and Molecular Biology and 2Center for Evolutionary Genetics,
c/o Department of Genetics and Molecular Biology, Università
‘La Sapienza’, Rome and 3Department of Biology, Università ‘Roma Tre’,
Rome, Italy
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 cytokinesisblocked 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.
Introduction
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
4To
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 interchromosomal, 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.
Materials and methods
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
whom correspondence should be addressed. Tel: 139 06 49902762; Fax: 139 06 49387139; Email: acarere@iss. it
© UK Environmental Mutagen Society/Oxford University Press 1999
491
A.Carere et al.
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 antirabbit antibodies. DNA was counterstained with 1 µg/ml 4,6-diamidino-2phenylindole 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).
Results
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
492
Fig. 1. Frequency of (A) chromosome non-disjunction (unbalanced
distribution of signals in the main nuclei of binucleated cells),
(B) chromosome loss (signals present in micronuclei) and (C) chromosome
hyperploidy (binucleated cells with both nuclei hyperploid) in the study
population (24 subjects). Each box represents the inter-quartile range of
frequencies with the bold line showing the median value. Vertical lines
show the range of values which fall within 1.5 box lengths, open circles
show outlier values falling between 1.5 and 3 box plot lengths and asterisks
show extreme values falling outside 3 box plot lengths.
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 malsegregation in lymphocytes
Fig. 3. Relationships between ageing and incidence of malsegregation of
pooled autosomes (chromosomes 7, 11 and 18). (A) Non-disjunction
(Spearman correlation coefficient 5 0.342, P 5 0.051, equation for linear
regression y 5 0.12x 1 1.09) and (B) hyperploidy (Spearman correlation
coefficient 5 0.321, P 5 0.063, equation for linear regression
y 5 0.10x – 0.92).
Fig. 2. Relationships between ageing and frequency of (A) chromosome X
non-disjunction (Spearman correlation coefficient 5 0.433, P 5 0.017,
equation for linear regression y 5 0.11x – 0.53), (B) chromosome X loss
(Spearman correlation coefficient 5 0.326, P 5 0.060, equation for linear
regression y 5 0.06x – 0.27) and (C) chromosome X hyperploidy
(Spearman correlation coefficient 5 0.547, P 5 0.003, equation for linear
regression y 5 0.06x – 1.32). Overlapping data are presented as circles.
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).
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 5
0.017 and 0.003, respectively) and close to significance for
chromosome loss (P 5 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 5 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 5 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 5 0.009), with no significant association
with the age of subjects (data not shown).
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 5 0.002;
Figure 4A) and for the pooled autosomes (P 5 0.036; Figure
4B). Considering each autosome separately, a positive correlation between in vitro and in vivo malsegregation was observed
for chromosome 18 (P 5 0.021) and a tendency toward
significance for chromosomes 7 and 11 (P 5 0.092 and 0.158,
respectively).
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A.Carere et al.
Table I. Incidence of single and multiple malsegregation events in cultured
human lymphocytes, as detected by FISH with centromeric probes for two
different chromosome pair mixes
Malsegregation eventsa
Observed
Fig. 4. Relationships between frequencies of in vitro non-disjunction and
in vivo malsegregation (hyperploidy) of (A) chromosome X (Spearman
correlation coefficient 5 0.577, P 5 0.002, equation for linear regression
y 5 0.28x 1 0.03) and (B) pooled autosomes (chromosomes 7, 11 and 18)
(Spearman correlation coefficient 5 0.374, P 5 0.036, equation for linear
regression y 5 0.25x 1 1.88). Overlapping data are presented as circles.
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.
Discussion
In this study the inter-individual variation in potential biomarkers for genomic instability was investigated in cytokinesisblocked 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 mal494
Expectedb
Chromosome X-18 mix (48 000 cells scored)
Singlea
Ch X
288
Ch 18
38
Total
326
Multiplea
Ch X-18
27
Ch18-18
10
Total
37
0.56
0.04
0.6
Chromosome 7–11 mix (23 900 cells scored)
Singlea
Ch 7
52
Ch 11
69
Total
121
Multiplea
C 7–11
2
Ch 7–7
2
Ch 11–11
6
Total
10
0.20
0.01
0.07
0.31
aSingle and multiple malsegregation events are classified as described in
Materials and methods.
bExpected values are calculated according to the hypothesis of chromosome
independence as follows. For multiple events involving two different
chromosomes (Ch 1 and Ch 2), frequency of malsegregation of
Ch 13frequency of malsegregation of Ch 23number of cells scored; for
multiple events involving two homologues of the same chromosome (Ch 1),
(0.53frequency of malsegregation of Ch 1)23number of cells scored.
Frequency of malsegregation of individual chromosomes were calculated as
follows. For chromosome X, total number of observed malsegregation
events/number of scored cells; for autosomes, (number of events involving
one homologue 1 23number of events involving both homologues)/number
of cells scored.
segregation) 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
Chromosome malsegregation in lymphocytes
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 nondisjunction 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 suscepti-
bility 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.
Acknowledgements
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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Received on February 2, 1999; accepted on June 7, 1999
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