Mutagenesis, Vol. 17, No. 6, 509-521,
November 2002
© 2002 UK Environmental Mutagen Society/Oxford University Press
Detection and characterization of mechanisms of action of aneugenic chemicals
Centre for Molecular Genetics and Toxicology, School of Biological Sciences, University of Wales Swansea, Singleton Park, Swansea SA2 8PP, UK
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Abstract |
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 Top Abstract IntroductionIdentification of aneugensThe classification of...The detection of non-disjunction...Thresholds of aneugenic activityMechanisms of actionMechanisms maintaining the...References |
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A comprehensive evaluation of the genotoxic potential of chemicals requires the assessment of the ability to induce gene mutations and structural chromosome (clastogenic activity) and numerical chromosome (aneugenic activity) aberrations. Aneuploidy is a major cause of human reproductive failure and an important contributor to cancer and it is therefore important that any increase in its frequency due to chemical exposures should be recognized and controlled. The in vitro binucleate cell micronucleus assay provides a powerful tool to determine the ability of a chemical to induce chromosome damage. The application of an anti-kinetochore antibody to micronuclei allows their classification into kinetochore-positive and kinetochore-negative, indicating their origin by aneugenic or clastogenic mechanisms, respectively. The availability of chromosome-specific centromere probes allows the analysis of the segregation of chromosomes into the daughter nuclei of binucleate cells to evaluate chromosome non-disjunction. Quantitative relationships between the two major causes of aneuploidy, chromosome loss and non-disjunction, can be determined. The mechanisms leading to chromosome loss and non-disjunction can be investigated by the analysis of morphological and structural changes in the cell division apparatus by the application of specific stains and antibodies for various cell division components. We illustrate such analyses by the demonstration of the interaction of the monomer bisphenol-A with the centrosome of the mitotic spindle and the folic acid antagonist pyrimethamine with the centromeres of chromosomes. Both types of modifications lead to the induction of aneuploidy in exposed cells. Our studies also implicate the products of the p53 and XPD genes in the regulation of the fidelity of chromosome segregation at mitosis.
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Introduction |
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TopAbstractIntroductionIdentification of aneugensThe classification of...The detection of non-disjunction...Thresholds of aneugenic activityMechanisms of actionMechanisms maintaining the...References |
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Genetic change can be produced by gene mutations, by structural chromosomal changes and by numerical chromosome changes (aneuploidy). The role of genetic changes in carcinogenesis has now been established via an extensive range of studies (for reviews see Barrett, 1995
; Duesberg et al., 1999; Cowell, 2001). The possible role of chemicals in the induction of both cancer-related genetic changes and genetic changes implicated in human birth defects has stimulated the development of a wide range of testing methods capable of detecting and assessing the genotoxic potential of chemicals (for a review see Phillips and Venitt, 1995).
The recognition of the genotoxic potential of a range of chemicals and the availability of validated test methods has led to the development of regulatory requirements in many countries which require the assessment of chemicals for which human exposure may occur (for a review see Carere et al., 1995). Thus, selected batteries of test methods are required to be used in many countries to assess the ability of chemicals to induce gene mutations and structural chromosome changes. However, until recently, testing strategies have not included a specific requirement to determine the potential of chemicals to induce aneuploidy, i.e. aneugenic activity (Aardema et al., 1998).
In view of the role played by aneuploidy in carcinogenesis and in human reproductive failure, this omission is perhaps surprising. It has been estimated that at least 50% of human conceptuses are aneuploid, although the vast majority are lost by miscarriage (Boué et al., 1975). Boveri (1914) had suggested a relationship between numerical aberrations and cancer as early as 1914. The observation of extensive aneuploidy in cancer cells (Mortens et al., 1997) indicates that modifications of the fidelity of chromosome segregation leading to karyotype instability may be critical events in the aetiology of at least a proportion of human tumours. Chromosome instability at an early stage in cancer progression may act as a driving force leading to the alteration in the copy number of one or more genes controlling cellular growth, thus affecting the expression of oncogenes and/or tumour suppressor genes.
The determination of the DNA reactivity of newly developed chemicals has been a regulatory requirement within the European Union for the past 20 years. Recent reassessments of the strategies for the testing of chemicals within the European Union has resulted in the increasing recognition of aneuploidy as an important end-point to consider when evaluating potential genotoxicity to both somatic and germ cells. The UK Department of Health's Advisory Committee on the Mutagenicity of Chemicals (COM) has recently recommended a requirement for the measurement of aneugenic potential in its revised guidelines for the testing of chemicals (Committee on the Mutagenicity of Chemicals, 2000) and it is expected that similar requirements will be introduced throughout Europe.
After the United Kingdom Environmental Mutagen Society (UKEMS) mutagenicity testing guideline recommendations were revised in 1993 (Kirkland and Fox, 1993), the UKEMS established a working group to review the field of chemically induced aneuploidy and to make recommendations concerning the most appropriate procedures to use for the detection of aneugens. The assays available at that time were examined in order to determine what was available and what aspects should be borne in mind when attempting to measure aneuploidy without dictating exact protocols (Parry,E.M. et al., 1995). The coincidence of the 25th meeting of the UKEMS with the International Workshop on Genotoxicity Testing (IWGT) this year in Plymouth makes this an ideal time to review the position with regard to aneuploidy detection and to consider what developments have occurred since 1995.
The development of appropriate methods for the detection and assessment of aneugenic chemicals has been a major thrust of a number of research programmes of the European Union (reviewed by Parry and Sors, 1993; Parry et al., 1996). These projects have progressed since that time to develop and to apply a range of methodologies capable of detecting and assessing the significance of aneugenic activity to both somatic and germ cells by the use of both in vitro and in vivo tests. A key procedure recommended by both the COM and the European Union Research Group is the in vitro micronucleus assay using the actin polymerization inhibitor cytochalasin B. We will concentrate our efforts in this review on this method and illustrate how the basic method may be used to identify aneugens and modified to assist in the characterization of modes of action of aneugenic chemicals.
We aim to review the detection of chromosome malsegregation in somatic cells and the potential origins and consequences of the events that lead to aneuploidy following errors in mitotic cell division.
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Identification of aneugens |
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TopAbstractIntroductionIdentification of aneugensThe classification of...The detection of non-disjunction...Thresholds of aneugenic activityMechanisms of actionMechanisms maintaining the...References |
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Aneuploidy is defined as a change in chromosome number from the normal diploid or haploid number for the species in question other than an exact multiple of the haploid number (polyploidy). It can be induced by chemical action upon a variety of cellular targets involved in cell division, as well as the chromosomes themselves (reviewed by Parry and Parry, 1989
), consequently its induction may not always correlate with gene mutation or chromosome aberration induction, both of which have DNA as their predominant target for chemical action. There are two main processes that give rise to aneuploidy: non-disjunction of chromosomes at anaphase so that one daughter cell becomes trisomic and the other becomes monosomic; chromosome loss during cell division so that one daughter cell becomes monosomic and the other one remains normal. In the latter case the lost chromosome may form a micronucleus that can be detected cytogenetically. Alternatively, it may be randomly re-incorporated into either one of the daughter nuclei so that either one daughter cell becomes trisomic and its partner monosomic or both daughter cells become diploid and the aneugenic event is effectively erased.
Experimental systems to detect aneuploidy must therefore be able to measure these events. They achieve this mainly by either counting chromosomes or identifying micronuclei containing whole chromosomes. Chromosome counting methods are usually performed on metaphase cells whereas micronuclei are examined in interphase cells. Problems associated with chromosome counting methods include the need to analyse metaphase cells from the second mitosis after treatment and the identification of chromosomes. These issues were discussed by Parry,E.M. et al.(1995) and there is little evidence in the literature since that time for the extensive use of metaphase analysis to detect aneugens.
In contrast, the in vitro micronucleus assay has undergone extensive study and development during this same period. The standard in vitro micronucleus (MN) assay can use many different cultured mammalian cell types to detect both clastogenic and aneugenic mutational events, and its history has been reviewed by Evans (1997). In order to count MN induced by chemical treatment, it is necessary to identify those cells that have divided once. This is achieved most commonly by the use of the actin polymerization inhibitor cytochalasin B (Carter, 1967; Maclean-Fletcher and Pollard, 1980; Fenech and Morley, 1985). This assay has been used for both the in vitro testing of chemicals and for biomonitoring studies using human lymphocytes. An international collaborative study, HUMN (Human MicroNucleus), was established in 1997 to evaluate the application of the MN assay to study human populations (Surralles and Natarajan, 1997; Fenech et al., 1999; Bonassi et al., 2001). A database of about 7000 subjects from 25 laboratories has been compiled and analysed to investigate and assess the effect of protocol variation, age, gender and any other identifiable variable on baseline MN frequency. Other studies have evaluated the performance of the MN assay for the detection of chemically induced genotoxicity in vitro (Matsushima et al., 2000; von der Hude et al., 2000; an ongoing interlaboratory study by Societe Francaise de Toxicologie Genetique). The last review of the in vitro MN assay by the IWGT took place at the Washington International Workshop on Genotoxicity Test Procedures 1999 (Kirsch-Volders et al., 2000). This was the first time that international experts from academic, regulatory and industrial backgrounds gathered to discuss a MN protocol to detect both clastogens and aneugens. An informal workshop also took place in Shizuoka in 2001 before the next IWGT meeting in Plymouth 2002, when international harmonization of the assay will be discussed further. The COM have included the in vitro MN test for clastogenicity and for indications of aneugenicity in their Stage 1 screening guidance document (Committee on the Mutagenicity of Chemicals, 2000), which indicated that they considered that the in vitro MN test was a suitable alternative to the in vitro chromosome aberration assay for the detection of both clastogenic and aneugenic potential.
The use of MN as a measure of chromosome damage in human lymphocytes was first proposed by Countryman and Heddle (1976) and subsequently modified by the inclusion of the cytokinesis blocking step, to identify cells that have undergone division, by Fenech and Morley (1985). It has attracted attention because of its potential simplicity, its ability to detect clastogens and aneugens, its ability to provide mechanistic information (Figure 1) and its potential for automation. We are concerned here with the use of this assay to detect aneugens, for which purpose it is essential to be able to detect the relationships between daughter cells from a single mitotic division in order to visualize and measure non-disjunction of chromosomes. Currently, the most extensively validated method to achieve this end-point is the binucleate micronucleus assay (BNMN) employing cytochalasin B to prevent cytokinesis (Figure 1).
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The in vitro micronucleus assay
The first consideration when using the in vitro MN assay concerns the potential application of cytochalasin B to produce binucleate cells. Table I
illustrates the data obtained in the MN assay using the rat cell line 208F exposed to colcemid in both the presence and absence of 6 µg/ml cytochalasin B. The data in Table I demonstrate that significant increases in MN were detected in mononucleate cells in the absence of cytochalasin B at colcemid concentrations 
17.5 ng/ml (~3 times induction). In contrast, in the presence of cytochalasin B there were significant increases in MN induction in binucleate cells at concentrations of 12.5 ng/ml (~5–9 times induction). In the presence of cytochalasin B a measure of the toxicity of colcemid was provided by the dose-dependent reduction in the proportion of binucleate cells.
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Cell strains and types may vary in their sensitivity to cytochalasin B but the concentration used is usually between 3 and 6 µg/ml. Therefore, an appropriate concentration should be used to suit the cell type. Different laboratories report binucleate cell frequencies in different cells of from 40 to 80% and there may be much inter-individual variation between human lymphocyte cultures (Bonassi et al., 2001
). The influence of cytochalasin B concentration on the frequency of binucleate cells and MN can be seen in Table II for the human lymphoblastoid cells AHH-1 and MCL-5. Both cell strains had similar sensitivity to cytochalasin B and in our laboratory we have selected 3 µg/ml as a suitable cytochalasin B concentration for routine use.
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The classification of micronuclei |
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TopAbstractIntroductionIdentification of aneugensThe classification of...The detection of non-disjunction...Thresholds of aneugenic activityMechanisms of actionMechanisms maintaining the...References |
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The mechanism of MN induction can be characterized as clastogenic and/or aneugenic by the detection of the absence or presence of kinetochore protein within the MN (Parry,J.M. and Parry, 1987
; Eastmond and Tucker, 1989; reviewed by Kirsch-Volders et al., 1997). This classification method uses anti-kinetochore antibody staining to identify the presence of kinetochore protein associated with the centromeres of chromosomes or its absence from acentric chromosome fragments (Brinkley et al., 1985). Secondary antibodies can be used to amplify the signal of the first bound antibody and the background chromosomes can be counterstained with DAPI for contrast. The cells are examined and scored under a fluorescence microscope. This classification system is based upon the association of kinetochores with centromeres and the concept that a centric chromosome or centric chromosome fragment in a MN has arisen from an aneugenic event. However, kinetochore damage may be a potential aneugenic mechanism, therefore, an alternative method to identify centromeres is to employ fluorescently labelled centromeric DNA probes. This has become possible due to the development of repetitive DNA sequence probes for centromeric and pericentromeric regions [for human and rat (van Goethem et al., 1995; de Stoppelaar et al., 2000) and mouse chromosomes (Weier et al., 1991; Boei et al., 1994); reviewed by Eastmond et al. (1995)]. Russo et al. (1996a) have employed the primed in situ DNA synthesis (PRINS) technique to mark centromeres and telomeres in mouse cells.
In Tables III–V we illustrate both the suitability of the in vitro MN assay for use with a range of cell types, i.e. human fibroblasts, human lymphoblastoid cells and Chinese hamster V79 cells, and the use of anti-kinetochore antibody labelling to distinguish between MN containing acentric fragments and those containing whole chromosomes. Table III illustrates the comparative results obtained when a primary human fibroblast culture was exposed to three known aneugens, vinblastine, griseofluvin and colcemid, and a clastogen, mitomycin C. The data demonstrate that all four compounds induced dose-dependent increases in MN. However, all three aneugens induced increasing frequencies of kinetochore-positive MN, whereas the clastogen, mitomycin C, induced increasing frequencies of kinetochore-negative MN. This approach can be extended to investigate and compare the effects of metabolically linked chemicals such as ethyl alcohol and acetaldehyde using MCL-5 cells, which have some metabolic competence (Table IV). Whereas ethyl alcohol induced primarily kinetochore-positive MN, acetaldehyde induced more kinetochore-negative MN. Another investigation that distinguishes the mechanisms of action of etoposide and podophylotoxin in MN induction in V79 cells is shown in Table V. Etoposide induced mainly chromosome breakage events whereas podophyllotoxin was a specific inducer of aneuploidy.
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Baseline values of kinetochore-positive MN show some variation between cell cultures and cell types from ~40 to 70%, as can be seen in the tables shown here.
It is often assumed that all chromosomes are equally sensitive to the effects of aneugenic chemicals and that any differences in response may be accounted for by other factors, such as size-related effects on the dynamics of cell division or selection. Aspects of chromosome structure that might possibly influence their segregation during cell division are their length, centromere position, chromatin structure, centromere size and associated hetreochromatin. In addition to the DNA probes that detect all centromeres, human chromosome-specific centromere probes now have wide commercial availability and may be used to investigate chromosome-specific loss into MN. Hando et al. (1994) and Catalan et al. (1995) used such probes to demonstrate the preferential loss of chromosome X into MN associated with female age.
The induction of chromosome-specific responses by aneugenic chemicals is a little studied field, but human chromosome-specific probe availability and the BNMN assay make this a feasible proposition. Caria et al. (1996) reported that acrocentric chromosomes are preferentially sensitive to the aneugenic effect of colchicine. A similar effect was found by Migliore et al. (1995) in MN induced by vanadium salts. Fimognari et al. (1997) studied the loss of chromosomes 1, 7, 11, 14, 17 and 21 into MN in human lymphocytes exposed to ionizing radiation. The results support a random model of radiation-induced aneuploidy. Wuttke et al. (1997) also found no difference in the frequency of chromosome 2 or 7 loss into MN after exposure to X-rays, however, chromosome 7 showed an elevated frequency after colchicine treatment. Guttenberg and Schmid (1994) reported that chromosomes 1, 9, 15, 16 and Y were preferentially lost into MN after 5-azacytidine exposure, although this result was not confirmed by Cimini et al. (1996). Chung et al. (2002) have compared the loss of chromosomes 7 and 8 into MN induced by exposing human lymphocytes to the benzene metabolite 1,2,4-benzenetriol and found that loss of chromosome 8 was more frequent than that of chromosome 7, although both chromosomes showed a dose-related response.
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The detection of non-disjunction in the in vitro binucleate micronucleus assay |
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TopAbstractIntroductionIdentification of aneugensThe classification of...The detection of non-disjunction...Thresholds of aneugenic activityMechanisms of actionMechanisms maintaining the...References |
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The availability of human chromosome-specific probes now means that chromosome non-disjunction can be examined in the BNMN assay (Figure 1
). Using this method of marking chromosomes, in addition to the system described above to classify MN, it is possible to detect both chromosome loss and chromosome non-disjunction in human cells (Zijno et al., 1994). Russo et al. (1996b) have described the use of PRINS to evaluate chromosome loss and non-disjunction in mouse splenocytes in a BNMN assay. The ability to detect FISH signals in both macro- and micronuclei in a cell that has been through mitosis represents the major improvement to the study of aneuploidy since 1995. This approach is dependent upon probe availability and thus largely limited to human cells.
Tables VI and VII provide examples of the data obtained using this method to detect both chromosome loss and non-disjunction in primary human fibroblasts after exposure to diethylstilboestrol. Here the spontaneous levels of chromosome loss were 0.56% (56% of 1) and of non-disjunction were 11.5% (when the frequency of non-disjunction for three measured chromosomes is adjusted to what might be expected for the set of 23 chromosomes assuming random participation). Thus, non-disjunction occurred at a 20-fold greater frequency than chromosome loss spontaneously in this cell culture. After exposure to diethylstilboestrol the ratio chromosome loss:non-disjunction was 2:30 at 5000 µg/ml (non-disjunction 15x chromosome loss) and 2:206 at 10 000 µg/ml (non-disjunction 100x chromosome loss). No differences could be detected between the rates of non-disjunction of chromosomes 10, 17 and 18 after these exposures. Zijno et al. (1994) estimated that the spontaneous frequencies of chromosome loss and non-disjunction for chromosome X in lymphocytes from normal male and female donors were 0.25 and 0.3 (non-disjunction) and 0.05 and 0 (chromosome loss), respectively.
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Thus, these studies all support the observation that non-disjunction is the major mechanism of aneuploidy production. Other studies examining induced chromosome loss and non-disjunction indicate that non-disjunction is detectable after a lower exposure. Kirsch-Volders et al. (1996)
examined the relative induction of chromosome loss and non-disjunction by ionizing radiation by the BNMN assay. Comparing this method with the analysis of metaphase cells by whole chromosome painting of the same chromosomes, they found that non-disjunction in the BNMN gave the highest rates. Marshall et al. (1996) reported similar findings after treatment with the aneugens colchicine, vinblastine and carbendazim. Similar findings have been reported by Zijno et al. (1996). |
Thresholds of aneugenic activity |
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TopAbstractIntroductionIdentification of aneugensThe classification of...The detection of non-disjunction...Thresholds of aneugenic activityMechanisms of actionMechanisms maintaining the...References |
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Theoretically, it is to be expected that certain types of aneuploidy, where multiple targets are involved (e.g. the mitotic spindle), should have a thresholded dose–response curve of induction. This type of response would have implications for any risk assessment considerations (Parry,J.M. et al., 1994
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One advantage of the BNMN assay is that large numbers of cells can be examined and the method has the potential for adaptation to automated analysis. A number of laboratories have investigated the use of flow cytometry to provide rapid counts on large cell numbers. It appears that this approach is probably more suited to the simple MN test or the erythrocyte MN assay, where a fairly simple image is being recognized. A recent report describes a method of measuring micronucleated erythrocytes in human blood that identifies early cells before the MN have been removed by the spleen showing potential for automation and assessment of human exposure (Dertinger et al., 2002).
The analysis of large numbers of cells allows the required sensitivity to investigate the kinetics of aneuploidy induction and the determination of thresholds of activity (Elhajouji et al., 1995, 1997; Marshall et al., 1996; Bentley et al., 2000).
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Mechanisms of action |
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TopAbstractIntroductionIdentification of aneugensThe classification of...The detection of non-disjunction...Thresholds of aneugenic activityMechanisms of actionMechanisms maintaining the...References |
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The CBMN assay measures both chromosome loss and non-disjunction, allowing comparisons between these two events to be made and studied and to aid in the identification of the mechanisms of aneuploidy induction.
As discussed earlier, the concept of a potential threshold of action of aneugenic chemicals has thus far been considered only for those chemicals which function by modifying the assembly or functioning of the cell division spindle (see for example Committee on the Mutagenicity of Chemicals, 2000). As we have illustrated here, the BNMN assay is a valuable tool for identifying the ability of a chemical to induce chromosome loss and non-disjunction. However, the classification of the mechanism of action of aneugens requires the use of other methodologies, as we illustrate in this section.
The European Union study on the detection of aneugenic chemicals (Parry et al., 1996) evaluated the in vitro activity of the folic acid antagonist pyrimethamine which suggested that the chemical was capable of inducing MN in the MN assay, as shown in Table VIII. To investigate the potential cellular target(s) of pyrimethamine, we undertook an analysis of the morphology of cultured Chinese hamster cells during mitotic cell division and chromosome segregation in the presence of pyrimethamine. In this study cells were exposed to 100 µg/ml pyrimethamine for one cell cycle and then fixed at time intervals of up to 12 h. Following fixation, the cells were stained with safranin O to visualize chromosomes and brilliant blue R to visualize the spindle structure (Parry et al., 1982). Table IX illustrates the results of the pyrimethamine cell morphology study. In this table we illustrate the frequencies of cells in which chromosomes could be observed dislocated from the metaphase plate and/or lagging during anaphase and telophase. Such data suggest that the MN observed in the binucleate cell study are the result of chromosome loss from the metaphase plate. However, cells exposed to pyrimethamine were observed to have apparently normal mitotic spindles and there was no evidence of any recovery of cells from the damage which produced chromosome dislocations. Unlike chemicals classified as spindle inhibitors, there was no evidence of the production of C mitosis in which the chromosomes were blocked at metaphase and scattered throughout the cell in the absence of an intact spindle (Warr et al., 1993) except for the positive control treatment with 0.02 µg/ml colcemid.
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To further investigate the mechanism of pyrimethamine-induced aneuploidy we undertook a chromosome number study using primary Chinese hamster LUC2 cells (Warr et al., 1993
). In this study the cells were exposed to a dose range of 0–100 µg/ml pyrimethamine, followed by exposure to colcemid, and the resultant metaphase cells were analysed for chromosome number in cells where the cell membrane was intact, to reduce artefactual chromosome loss (Danford, 1984). The data obtained from the chromosome analyses are shown in Table X. The results demonstrate a dose-dependent increase in cells with a chromosome number lower than the 22 chromosome karyotype of the Chinese hamster, i.e. monosomic cells. With the exception of the dose of 50 µg/ml, there was no evidence of an increase in trisomic cells. Further analysis of the cells exposed to pyrimethamine indicated the presence of cells containing B group chromosomes which were broken at their centromeres. Cells containing centromere-damaged chromosomes were evaluated and were classified as examples of `segmental aneuploidy'. The results shown in Table X illustrate that pyrimethamine induced dose-dependent increases in segmental aneuploidy which was significant at doses >2.5 µg/ml. We provisionally conclude from this study that the primary mechanism of action of pyrimethamine is the induction of damage to centromeric DNA which results in a failure of attachment of such damaged chromosomes to the mitotic spindle. We conclude that chromosomes with damaged centromeres fail to correctly attach to the spindle, thus producing MN and monosomy. Renzi et al. (1996) and Eastmond et al. (1995) have demonstrated a similar mechanism of action for mitomycin C.
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To further demonstrate the progression from the detection of aneugenic activity to identifying the potential cellular targets of aneugenic activity we illustrate here our studies on the chemical bisphenol-A. This substance is used as a monomer in the manufacture of polycarbonate and epoxy resins. The main toxicological interest in bisphenol-A has been due to its oestrogenic activity in vitro and in vivo (Schafer et al., 1999
; Ema et al., 2001). Bisphenol-A was evaluated for its ability to induce MN in the human lymphoblastoid cell line MCL5. As shown in Table XI, bisphenol-A induced a dose-dependent increase in MN which was significant at concentrations >10 µg/ml. The MN were treated with an anti-kinetochore antibody and classified for the presence or absence of kinetochore signals. The data in Table XI demonstrate that the relative proportions of kinetochore-positive MN increased from 34–38% in the controls to 63% at the bisphenol-A concentration of 20 µg/ml. Such an increase in the relative frequency of kinetochore-positive MN indicates that bisphenol-A is capable of inducing chromosome loss and can thus be provisionally classified as an aneugen.
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To confirm the aneugenic potential of bisphenol-A we analysed the mitotic segregation patterns of chromosomes 8, 17 and 20 in the main daughter nuclei of MCL5 cells exposed to concentrations of bisphenol-A up to 20 µg/ml in the BNMN test. The data obtained (Table XII
) demonstrate that bisphenol-A induced a dose-dependent increase in non-disjunction of chromosomes 8, 17 and 20. Thus, this study confirms our provisional classification of bisphenol-A as an aneugenic chemical.
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To investigate the potential cellular targets, damage to which by bisphenol-A may lead to the production of aneuploid cells, we initially examined the fidelity of mitotic spindle formation and chromosome behaviour at cell division in bisphenol-A-exposed Chinese hamster V79 cells. In this study cells were exposed to bisphenol-A for one cell cycle and then stained with safranin O and brilliant blue R to visualize the chromosomes and spindle, respectively (Parry,E.M. et al., 1982
). Cells were classified with regard to the presence of abnormal metaphases and aberrations of any of the stages of mitosis. The data (Table XIII) demonstrate a dose-dependent increase in the frequencies of both abnormal metaphases and all mitotic aberrations. Individual cells showed a variety of aberrations, with the common presence of cells with abnormalities of the spindle poles, frequently being multipolar (see Figure 2).
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The observation of multipolar spindle structures in bisphenol-A-treated cells suggests that the chemical may interact with components of the centrosomes which make up the poles of the mitotic spindle. To investigate this potential mechanism we treated the bisphenol-A-exposed cells with antibodies to
-tubulin and 
-tubulin, which interact with the microtubules of the mitotic spindle and the centrosomes, respectively (Schiebel, 2000). The two anti-tubulin antibodies were identified microscopically by the use of specific fluorochromes, i.e. a green fluorochrome for -tubulin and a red fluorochrome for -tubulin. The antibody-treated cells were then classified according to the types of spindles observed as normal bipolar and aberrant tripolar, tetrapolar or multipolar structures. The data obtained from the analysis of polar structure is illustrated in Table XIV. It demonstrates that bisphenol-A exposure resulted in significant increases in tripolar spindles at 14 µg/ml and tetrapolar and multipolar spindles at 28 µg/ml. Our data indicate that a critical target for the interaction of bisphenol-A whose modification leads to aneuploidy is the centrosome.
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We conclude that interactions of bisphenol-A with the centrosome may result in the production of mitotic cells with deviations from the normal bipolar structure. When the chromosomes of a cell with abnormal polar structures enter anaphase they may become abnormally segregated, leading to chromosome loss and non-disjunction and the subsequent production of aneuploid progeny. A number of authors have demonstrated that the correct functioning and number of centrosomes is critical to maintaining karyotype integrity (Khodjakov and Reider, 2001) and that abnormal centrosome function is important in cancer development and progression (Pluta et al., 1995
; Pihan et al., 1998; Brinkley, 2001). Factors such as the overexpression of cyclin E and the loss of p53 have been shown to lead to centrosome amplification (Fukasawa et al., 1996; Mussman et al., 2000). In view of the important role of the centrosome in ensuring the correct segregation of chromosomes, the capacity of a chemical such as bisphenol-A to modify centrosome behaviour can be considered an important factor in toxicological evaluation. |
Mechanisms maintaining the integrity of the karyotype |
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TopAbstractIntroductionIdentification of aneugensThe classification of...The detection of non-disjunction...Thresholds of aneugenic activityMechanisms of actionMechanisms maintaining the...References |
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There is an extensive range of evidence indicating that the activity of cellular checkpoint mechanisms is essential for the maintenance of the integrity of the genome (Hartwell and Kastan, 1994
). The p53 protein has been shown to function at the G2/M checkpoint (Stewart et al., 1995) and during the stages of formation of the cell division spindle (Cross et al., 1995). Previous studies in our laboratories (Parry et al., 1998) have demonstrated that defective functioning of the p53 protein leads to elevations in aneuploidy in cultured cells. To aid in the dissection of the cellular factors and activities which may influence the response of cells to aneugenic chemicals we have determined the influence of the XPD gene upon the sensitivity of cultured human cells to the aneugens colcemid and vinblastine.
Human fibroblast cultures, including the repair-proficient cell line HF12 and the XPD mutant cell cultures XP16BR and XP1BR, were analysed for their response to colcemid and vinblastine and the influence of the XPD gene upon the conversion of damage to the mitotic spindle into aneuploid progeny cells. In these experiments we exposed cells to either colcemid or vinblastine for one cell cycle. After washing, the treated cells were either cultured for a period of 10 h in the presence of 3 µg/ml cytochalasin B or cultured for one, two or three cell cycles before a 10 h culture period in the presence of 3 µg/ml cytochalasin B. The results of these studies are illustrated in Tables XV and XVI, which demonstrate that in the repair-proficient cell line, HF12, there was a significant increase in the induction of MN following both colcemid and vinblastine treatment, which were primarily kinetochore-positive and thus presumably the result of whole chromosome loss. However, when the cells were grown for up to three post-treatment cell cycles before cytochalasin B treatment there was a progressive reduction in the frequency of MN. Similar reductions in MN frequency with different harvesting time have been observed in human lymphocytes following vincristine treatment by Channarayappa et al. (1992).
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In marked contrast to the cell cycle-dependent reduction in induced chromosome loss in the repair-proficient culture, no such reduction could be observed in the XPD-defective cell lines following both colcemid and vinblastine treatment. The level of MN remained at a consistently high level irrespective of the number of cell cycles before fixation in the presence of cytochalasin B.
To further evaluate the influence of the XPD gene product upon induced aneuploidy we investigated the induction of non-disjunction by colcemid and vinblastine in both repair-proficient and XPD-defective cell lines. In these experiments the cell cultures were exposed to low doses of colcemid and vinblastine for a period of 10 h followed by a cell cycle in the presence of 3 µg/ml cytochalasin B. The distribution of chromosomes 16 and 18 in binucleate cells was assessed and the results are illustrated in Table XVII. The study demonstrated that at the colcemid and vinblastine concentrations used there was no increase in non-disjunction of chromosomes 16 and 18 in the repair-proficient HF12 cell line. In contrast, these low concentrations of colcemid induced significant increases in non-disjunction in both the XPD-defective cultures and by low concentrations of vinblastine in the XPD-defective mutant TTD1BEL.
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These data demonstrate that the XPD gene product plays a role in the events which protect human cells from the aneugenic effects of chemicals. We can postulate that the role of the XPD gene product relates to its role in the TF11H repair/transcription product (reviewed by Drapkin et al., 1994
). Presumably, a defect in TF11H-mediated transcription can reduce the ability of a cell to recruit tubulin subunits to chemically damaged spindles. We might also predict that the influence of the XPD gene product upon induced aneuploidy may also relate to the action of the TF11H complex upon p53-mediated checkpoint arrest (Jones and Wynford-Thomas, 1995).
In a recent publication, de Boer et al. (2002) have suggested that ageing in mice carrying the defective XPD gene found in human trichothiodystrophy (TTD) is caused by unrepaired DNA damage, compromised transcription and enhanced apoptosis. However, it is attractive to suggest that inadequately repaired spindle components and thus potential aneuploidy may also play a significant role in ageing.
The studies reviewed here indicate that chemical interactions with a number of cell components and activities, such as synthesis and functioning of the spindle fibres, the activity of the centrosome and modification of centromeres, may lead to chromosome loss and non-disjunction, events which may lead to the production of aneuploid progeny cells.
It is also becoming increasingly clear that aneuploidy is not an inevitable consequence of damage to the cellular components. Rather, the mammalian cell is capable of repairing and/or eliminating damaged cells before aneuploidy is produced. The demonstration of a role for the p53 and XPD gene products provides some clues to the mechanisms that may protect cells from potentially aneugenic damage.
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Acknowledgments |
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The studies of the authors were supported in part by funds from the European Union Environmental Programme, the UK Health and Safety Executive and the UK Food Standards Agency.
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Notes |
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1 To whom correspondence should be addressed. E.M.Parry{at}swansea.ac.uk
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Received on June 27, 2002; accepted on July 23, 2002.
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