Mutagenesis, Vol. 17, No. 1, 83-88,
January 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Influence of donor age on vinblastine-induced chromosome malsegregation in cultured peripheral lymphocytes
Laboratory of Comparative Toxicology and Ecotoxicology,Istituto Superiore Sanità, Viale Regina Elena 299, I-00161 Rome, Italy
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Abstract |
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The incidence of spontaneous aneuploidy in human somatic and germ cells is known to be positively associated with aging. However, the influence of age on the individual susceptibility to chemically induced chromosome malsegregation has not been elucidated. In this study the spindle poison vinblastine (VBL) was used as a model compound to assess the influence of donor age on chemically induced chromosome malsegregation in cultured lymphocytes. Blood cultures from 20 female donors belonging to two different age groups (10 <30 years and 10 >50 years) were treated with VBL (7.5 ng/ml) from 43 h after mitogen stimulation until harvest at 60 h, i.e. during the time interval corresponding to G2/M. In order to block cytokinesis, cytochalasin B (6 µg/ml) was added to cultures at 44 h. For each donor the incidence of micronuclei, polyploidy and malsegregation (non-disjunction and loss) of chromosomes X and 8 was determined using fluorescence in situ hybridization with chromosome-specific centromeric probes. Both background incidence of micronuclei and spontaneous chromosome X non-disjunction were significantly elevated in older donors. Individual responses to VBL treatment showed wide interindividual variability, which was not significantly associated with the age of the donor. In both age classes chromosome X was more susceptible than chromosome 8 to both spontaneous and VBL-induced malsegregation. These results indicate that donor age has a limited influence on the aneugenic effects exerted by VBL in peripheral lymphocytes in vitro. Other factors have to be considered to account for the large interindividual variation in sensitivity to VBL challenge observed in this work.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Aneuploidy has severe consequences on human health, at both the somatic (Sen, 2000
) and germ cell levels (Hecht and Hecht, 1987). Among factors affecting the fidelity of chromosome segregation, aging has a prominent role. In fact, both the incidence of aneuploidy in germ cells (Eichenlaub-Ritter, 1996; Shi and Martin, 2000) and the incidence of chromosome malsegregation in somatic cells in vitro (Nowinski et al., 1990) have been shown to be clearly associated with the age of the subjects under study.
In humans aging differentially affects the segregation of specific chromosomes, suggesting that chromosome-specific factors modulate segregation fidelity. Increased malsegregation of chromosomes X and 21 has been observed in peripheral lymphocytes (Nowinski et al., 1990) and in sperm (Downie et al., 1997) and offspring (Hassold et al., 1996; Hassold and Hunt, 2001) of aged subjects. In aged donors the sex chromosomes retain their proneness to malsegregate during lymphocyte growth in vitro (Stone and Sandberg, 1995), indicating that the relative greater propensity of the X chromosomes to malsegregate depends on intrinsic chromosome features.
As to the age-related factor(s) modulating the fidelity of chromosome segregation, very little is known. A reduction in the number of chiasmata has been postulated in the case of malsegregation of chromosome 21 at meiosis (Petersen and Mikkelsen, 2000). In addition, an impairment of centromere/kinetochore function has been invoked to explain the high spontaneous malsegregation of sex chromosomes in aged women (Nakagome et al, 1984; Hando etal., 1994; Catalán etal., 2000a). However, in view of the structural complexity and fine control network of the machinery of chromosome segregation (Kirsch-Volders et al., 1998), it is conceivable that multiple factors might contribute to the lower fidelity of chromosome segregation in aged subjects. Indeed, a recent analysis of gene expression in human fibroblasts, using microarray chips, highlighted down-regulation of multiple genes involved in cell cycle progression and mitosis control in aged individuals (Ly etal., 2000).
The influence of factors affecting spontaneous chromosome segregation on chemically induced aneuploidy is poorly understood, even though it is conceivable that an impairment of mechanisms controlling ploidy stability could equally affect spontaneous and induced aneuploidy. The elucidation of this aspect could provide relevant information on mechanisms controlling the fidelity of chromosome segregation and identify individual susceptibility factors relevant for chemical risk assessment.
In order to address some of the points raised above, in this study the influence of age on chemically induced malsegregation has been investigated. Vinblastine (VBL) was used as a model compound to assess the sensitivity to spindle poisons of peripheral lymphocytes of young (<30 years) and elderly (>50 years) female donors. The incidence of spontaneous and induced micronuclei and chromosome malsegregation was determined in cytokinesis-blocked binucleated lymphocytes. Through fluorescence in situ hybridization (FISH) with chromosome-specific centromeric probes, both chromosome loss and non-disjunction were simultaneously detected (Zijno etal., 1994). Chromosomes X and 8 were selected because of the frequent occurrence of sex chromosome trisomy in live aneuploids (Thomas et al., 2001) and of autosome 8 abnormalities in tumors (Pettenati et al., 1994; Satge et al., 2001).
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Materials and methods |
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Study population
Twenty healthy non-smoker female donors, with no recent diagnostic X-ray or occupational exposure to (geno)toxic agents, participated in the study. Ten donors were above 50 years old (`elderly' class, age range 51–63 years) and 10 below 30 years old (`young' class, age range 24–29 years). Blood samples (5 ml) were collected once by venipuncture in heparinized vacutainers. In order to account for experimental variability, donors were matched by age (i.e. each young subject was matched to an old one) in all experiments. All subjects were informed of the intent of the study and gave their consent.
Cell culture
Lymphocyte cultures were established with 0.5 ml of whole blood, added to 4.5 ml of RPMI 1640 medium (Gibco) with 25 mM HEPES buffer and L-glutamine, supplemented with 20% heat-inactivated fetal calf serum (Hyclone), 1% penicillin (5000 U/ml) and streptomycin (5000 µg/ml) solution (Flow) and 2% phytohemagglutinin (PHA) HA 15 (Murex), and incubated at 37°C. Cytokinesis was blocked with 6 µg/ml cytochalasin B (Sigma), added 44 h after PHA stimulation. Cells were harvested 60 h from the beginning of culture.
Chemical treatments
VBL (Sigma) was dissolved in distilled water to obtain a stock solution (50 µg/ml), which was aliquoted and stored at –20°C until use. For each experiment one aliquot of VBL was thawed and added to duplicate cell cultures 43 h after stimulation, at a final concentration of 7.5 ng/ml. This dose was selected on the basis of the results of a previous study (Zijno et al., 1996b), in order to elicit significant disturbance of mitotic segregation with no overt toxicity or mitotic arrest. Cultures were harvested at 60 h, lymphocytes pelleted by centrifugation (10 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). Fixed cells were dropped onto clean, wet slides. Slides were either stained with Giemsa or stored at –20°C for subsequent in situ hybridization.
Fluorescence in situ hybridization
FISH was performed on binucleated lymphocytes using commercial probes (ONCOR) for the alphoid sequences of human chromosomes X (probe DXZ1) and 8 (probe D8Z2). Molecular hybridization of the probes and slide pretreatment were carried out essentially according to the protocol provided by the manufacturer with the ONCOR Chromosome in situ Kit and as previously described (Zijno et al, 1996c). A mixture of chromosome X and 8 probes was prepared using a chromosome X probe labeled with digoxigenin and a biotinylated chromosome 8 probe diluted in Hybrisol VI (ONCOR) to a final concentration of 1 ng/µl. Detection of the biotin-labeled probe was performed with fluoresceinated avidin (FITC–avidin) (Vector). The fluorescence intensity was amplified with a biotinylated anti-avidin antibody (Vector), followed by an additional layer of FITC–avidin. The digoxigenin-labeled probe was detected using a rhodamine-conjugated sheep antidigoxigenin antibody (Boehringer Mannheim) followed by signal amplification with Texas Red (TR)-conjugated rabbit anti-sheep and goat anti-rabbit antibodies (Vector). DNA was counterstained with 4,6-diamidino-2-phenylindole (DAPI), at a concentration of 5 µg/ml in antifade solution.
Slide scoring
Giemsa stained slides were used for the analysis of nuclear division index (NDI), mitotic index (MI) and micronuclei. NDI was calculated as follows: [(1xM1) + (2xM2) + (3xM3) + (4xM4)]/1000, where M1, M2, M3 and M4 are mono-, bi-, tri- and tetra-nucleate cells, respectively, in 1000 scored cells (Eastmond and Tucker, 1989). MI was determined by scoring mitotic figures in 1000 cells for each experimental point. Micronuclei in 1000 binucleated cells were scored according to Fenech (1993). Slides hybridized with the two probes were analyzed under a Leica DM-RB microscope equiped with a triple bandpass filter (Chroma) for simultaneous detection of TR, FITC and DAPI. Malsegregation of hybridized chromosomes was confirmed by inspection under blue or green light filter blocks, in order to specifically visualize the FITC and TR signals, respectively. At least 1000 binucleated cells were scored per experimental point. On the basis of the distribution of centromere signals in the main nuclei and micronuclei, binucleated cells were classified as normal, when all signals were evenly distributed in the two daughter nuclei, chromosome loss, when one or more signals were found in the micronucleus, and non-disjunctional, when an unbalanced distribution of signals in the main nuclei was observed.
On the same hybridized slides, in most experiments, 1000 mononucleated cells were scored for identification of polyploid cells (i.e. cells containing four signals for each of the two chromosomes).
Statistical analysis
Spontaneous and induced incidences of genetic end-points in the two age groups were compared by two-tailed Student's t-test for independent groups. Within each group, spontaneous and induced effects and malsegregation of chromosomes X and 8 were compared by two-tailed Student's t-test for paired groups. Correlation analyses were performed with the two-tailed Pearson correlation coefficient.
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Results |
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Effects of age on lymphocyte proliferation, spontaneous chromosome malsegregation and micronuclei
Data on cell replication indices (NDI and MI) and on micronuclei, chromosome malsegregation and polyploidy in lymphocyte cultures from young (<30 years) and elderly (>50 years) donors are presented in Table I
. In untreated cultures no significant age-related difference was observed for both NDI and MI, indicating that aging did not affect the proliferation of peripheral lymphocytes in vitro. On the other hand, the background incidence of micronuclei and both chromosome X non-disjunction and loss were consistently higher among elderly subjects compared with young donors (P < 0.001, t-test for independent groups), confirming the well-known effect of aging on these end-points. At variance with chromosome X, no significant differences in the frequencies of spontaneous non-disjunction and loss of chromosome 8 were observed in the two age groups. The incidence of polyploidy, a possible hallmark of gross perturbation of mitosis, was nearly similar in the two study groups.
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Effects of VBL on cell proliferation and genetic end-points in the whole study population
Individual and aggregated data on the effects induced by VBL in lymphocyte cultures are also shown in Table I
. VBL treatment slightly depressed proliferation of lymphocytes and induced mitotic arrest, as indicated by the reduction in NDI and by the increase in MI. Micronuclei, polyploidy and non-disjunction of both chromosomes X and 8 were significantly increased by chemical treatments (P < 0.001, t-test for paired groups), whereas no significant induction of chromosome loss (either chromosome X or 8) was observed.
Effects of age on VBL-induced micronuclei and chromosome non-disjunction
The effects of VBL on cell proliferation indices and genomic stability in the two age groups are summarized in Table II, where group means of induced effects (background subtracted) are shown. On average, higher values of induced micronuclei and chromosome X non-disjunction were observed among elderly donors. However, due to large interindividual variation, such differences did not attain statistical significance. No distinct age-related difference was observed for the other end-points.
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In order to assess the influence of experimental variability on the results, the data were further analyzed with the Pearson correlation coefficient. No significant correlation was observed within data from the same experiment, indicating that experimental variability did not contribute to the dispersion of individual values.
For an easier comparison of spontaneous and induced values, also in relation to the effect of age, individual data on VBL-induced micronuclei and non-disjunction of chromosomes X and 8 are depicted in Figures 1–3. Here, for each subject the result obtained after VBL treatment is plotted against the corresponding spontaneous value. An inspection of Figures 1 and 2 clearly shows an effect of aging on the spontaneous incidences of micronuclei and non-disjunction of chromosome X, represented graphically by the separation of young and elderly subjects along the abscissa. On the other hand, no similar clear-cut separation is observed along the orthogonal axis, representing VBL-induced effects. In other words, aging clearly affected the spontaneous incidence of micronuclei and chromosome X non-disjunction, but had no similar influence on VBL-induced effects, which are scattered, largely independent of the age of the subjects. A different picture is observed in Figure 3, which shows data on non-disjunction of chromosome 8. In this case, both spontaneous and VBL-induced effects were unaffected by the age of the donors. In any case, no correlation was observed between spontaneous and induced values for any of the end-points considered.
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Comparison of spontaneous and induced malsegregation of chromosomes X and 8
The parallel analysis of malsegregation of chromosomes X and 8 performed in this study allowed a direct and reliable comparison of the susceptibility to spontaneous and chemically induced malsegregation of chromosome X and of an autosome of similar size. Such analysis, performed in the same cell populations, was not biased by experimental or interindividual variations which flaw the majority of studies on this subject.
In the whole study group (Table I) chromosome X proved to be significantly more prone to spontaneous non-disjunction and loss than chromosome 8 (P < 0.001 for both end-points). The same comparison within each age group confirmed the higher susceptibility of chromosome X, compared to the autosome, to spontaneous non-disjunction and loss in elderly subjects (P < 0.001) and to spontaneous loss in young donors (P < 0.05).
After VBL treatment non-disjunction of chromosome X was induced more frequently than non-disjunction of chromosome 8 in the whole study population (P = 0.003) (Table II), even if the difference only reached statistical significance in the elderly group (P = 0.017) and not in the young subjects (P = 0.089).
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Discussion |
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Genome stability in humans is known to be influenced by several factors, including age, gender and chromosome-specific features. However, the role of these factors on chemically induced malsegregation has not been elucidated. In this study the influence of donor age on VBL-induced chromosome malsegregation in peripheral lymphocytes was investigated. Twenty female donors belonging to two age classes, previously shown to be associated with significant differencies in spontaneous malsegregation rates (Zijno et al., 1996a
), participated in the study. Peripheral lymphocytes from whole blood cultures of each donor were challenged with VBL and induction of micronuclei and malsegregation (loss and non-disjunction) of chromosomes X and 8 were evaluated.
Spontaneous malsegregation rates observed in the study population confirmed the well-known positive association between age of donor and incidence of micronuclei (Fenech and Morley, 1985; Bolognesi etal., 1999) and chromosome X non-disjunction (Zijno et al., 1996a,1996c) and loss (Hando et al., 1994; Catalán etal., 1995, 2000a) in cultured peripheral lymphocytes. No age-related differences were observed in spontaneous malsegregation of chromosome 8, indicating that age-related instability may not be a general trait of all human chromosomes. Therefore, the differential susceptibilities of chromosomes X and 8 to age-related instability made these chromosomes suitable targets to reveal the possible synergism of aging with chemically induced malsegregation.
Treatment of stimulated lymphocytes with VBL affected cell cycle progression (as shown by the NDI and MI values) and significantly increased the overall frequencies of micronuclei and polyploidy. VBL also effectively induced non-disjunction of both chromosomes X and 8, whereas no parallel increase in chromosome loss was observed, confirming the finding that non-disjunction is the prevalent effect induced in binucleated cells by sub-toxic doses of spindle poisons (Zijno et al., 1996b,1996c). In this respect, it was proposed that the reduced distance between spindle poles in cytokinesis-blocked cells would facilitate the random incorporation of lagging chromosomes in daughter nuclei, thus increasing the overall non-disjunction rate (Minissi et al., 1999).
A comparison of the results obtained with subjects of different ages did not highlight significant age-related differences in the induction of micronuclei, non-disjunction and polyploidy after VBL treatment. On the other hand, large interindividual variation was observed, not accounted for by experimental variability, which suggests the existence of a factor(s) other than age which effectively modulates the individual response to aneugenic chemicals. This unidentified modulating factor(s) could differentially affect spontaneous and chemically induced malsegregation, as suggested by the lack of a correlation between rates of spontaneous and induced micronuclei and chromosome non-disjunction. In principle, interindividual variation in the sensitivity to VBL might be due to polymorphic expression of genes affecting drug uptake or metabolism and its interaction with cellular targets, as well as the genetic control of spindle integrity. The results of this study do not actually rule out the possibility that aging could also modulate the response to spindle poisons to a limited extent, but they demonstrate that other factors have a prominent role. In this respect, these findings are in agreement with the results of a recent survey on colchicine sensitivity in lymphocytes of a large study population (Odagiri etal., 1997), where a ratio of 1.4, comparable with that recorded in this study, was observed for micronucleus induction in subjects in the age range 51–70 years compared with 19–30 years. Large interindividual variations and no relationship between spontaneous and induced values were also observed in this study.
A comparison of the rates of malsegregation of chromosomes X and 8 in the same subjects highlighted the greater susceptibility of the sex chromosomes to both spontaneous and chemically induced non-disjunction, irrespective of the age of the donor. The relatively greater proness to malsegregation of chromosome X, compared with chromosome 8 or other autosomes, has already been observed in cytokinesis-blocked lymphocytes treated with benomyl and carbendazim (Bentley et al., 2000) or griseofulvin and vanadate (Migliore etal., 1999) and in cultured lymphocytes treated with 1,3-butadiene metabolites (Xi etal., 1997). So far, no plausible mechanistic explanation has been given for the preferential involvement of chromosome X in chemically induced non-disjunction and chromosome loss. Loss of kinetochore function (Nakagome etal., 1984; Catalán et al., 2000a), premature centromere division (Fitzgerald, 1993) or telomere shortening (Surrallès et al., 1999) have been proposed to explain the spontaneous, age-related instability of chromosome X. In view of the lack of a synergistic interaction between aging and chemical treatment, it is unlikely that these mechanisms play a role in the increased susceptibility of chromosome X to the effects of spindle poisons. The presence in female donors of an inactivated copy of this chromosome has also been proposed to explain its higher susceptibility to chemically induced malsegregation (Bentley etal., 2000). However, over-involvement in spontaneous aneuploidy of both X chromosomes is suggested by a similar occurrence of the active and inactive X chromosomes in micronuclei (Surrallés et al., 1996) and in anaphase lagging (Catalán et al., 2000b). Furthermore, preliminary results from a study on male donors confirm the higher susceptibility of the X chromosome to malsegregation after VBL treatment also in this gender (Leopardi et al., 1998). Therefore, other chromosome features have to be considered to explain the low fidelity of segregation of chromosome X. One of these is the distal position of laggards formed by the X chromosomes at anaphase (Catalán et al., 2000b), which could facilitate subsequent malsegregation.
In conclusion, the results of this study indicate that the age of the donor has a limited influence on the induction of chromosome malsegregation in peripheral lymphocytes after in vitro treatment with VBL. On the other hand, aging significantly affects spontaneous malsegregation and genome stability, even though in a chromosome-specific way. Chromosome-specific traits also seem to determine, in addition to age-related effects, sensitivity to spindle poisons.
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Acknowledgments |
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The collaboration of Mrs Daniela Ferrari (Istituto Superiore di Sanita') in the selection and recruitment of study subjects is acknowledged. This work was partially supported by the Italian Ministry of Environment (Project PR 22-IS).
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Notes |
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1 To whom correspondence should be addressed. Tel: 39 06 49902680; Fax: =39 06 49387139; Email: zijno{at}iss.it
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References |
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Received on July 18, 2001; accepted on September 26, 2001.
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