Mutagenesis, Vol. 15, No. 2, 155-164,
March 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
In vivo cytokinesis blocked micronucleus assay with carbendazim in rat fibroblasts and comparison with in vitro assays
Laboratory of Health Effects Research, National Institute of Public Health and the Environment, PO Box 1, 3720 BA Bilthoven, The Netherlands
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A successful in vivo application of the cytokinesis blocked micronucleus assay for the detection of aneuploidy induced by carbendazim (CARB) was carried out in the granuloma pouch assay. This was performed in two ways: (i) in vivo exposure of the skin fibroblasts to cytochalasin B (cytB) and CARB, by simultaneous injection of both substances into the pouch; (ii) in vivo exposure to CARB followed by in vitro culturing of the fibroblasts in the presence of cytB. Only the first assay was successful. Injection of cytB (with or without the test compound) into the pouch resulted in the induction of binucleate cells in vivo, up to a maximum of 5% at 1 mg cytB/pouch. After injection of CARB (0–50 or 0–10 mg/pouch) and cytB (1 mg) into the pouch, aneuploidy was determined in the isolated binucleate fibroblasts by fluorescence in situ hybridization with a general centromeric probe and combinations of chromosome-specific probes (19p + 19q, 4q + Yq). With all probes, the induction of chromosome loss and/or non-disjunction by CARB was very pronounced; at 10 mg CARB/pouch the total malsegregation frequency of chromosomes 4, 19 and Y was ~300/1000 binucleate cells. In an in vitro cytokinesis block assay with CARB (0–2.5 µg/ml) in primary skin fibroblasts the induced aneuploidy frequencies were as high as observed in the in vivo assay. The use of two probes for chromosome 19, which enabled the scoring of chromosome breaks in addition to aneuploidy, revealed no significant induction of chromosome breaks by CARB. The frequency of polyploid mononucleate and binucleate cells was decreased after CARB treatment, in both the in vivo and in vitro assays. However, in an additional in vitro assay without cytB a major induction of polyploidy from 2.5 µg/ml CARB and above was observed, showing that cytB may interfere with polyploidy induction.
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Introduction |
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Numerical chromosome aberrations (aneuploidy or polyploidy) have major consequences for human health. Almost 50% of spontaneous abortions are aneuploid or polyploid and the incidence of numerical aberrations in live-born young is ~5/1000 births (Abruzzo and Hassold, 1995
; Hassold et al., 1996). In somatic cells, aneuploidy can play a role in malignant transformation, genetic instability and tumorigenesis (Oshimura and Barret, 1986; Li et al., 1997; Duesburg et al., 1998). Certain chemicals, such as colchicine and the fungicide carbendazim, are able to induce aneuploidy by interacting with the spindle apparatus. They bind to tubulin and prevent the formation of microtubules, thereby interfering with normal chromosome segregation (Davidse and Flach, 1977; Albertini et al., 1988; Parry,J.M. and Sors, 1993). Considering the consequences of aneuploidy for human health, efforts have been undertaken to develop screening tests and testing strategies for the detection of aneuploidy (Liang and Brinkley, 1985; Adler and Parry, 1993; Parry,E.M. et al., 1995; Parry,J.M. et al., 1996).
The in vitro cytokinesis blocked micronucleus assay [first described by Fenech and Morley (1985)] in combination with fluorescence in situ hybridization (FISH) has become a widely used method for the detection of aneuploidy (Boei and Natarajan, 1995; Kirsch-Volders et al., 1997; Surrallés and Natarajan, 1997). It allows the simultaneous determination of aneuploidy and chromosome breaks and is a likely candidate to replace the conventional in vitro chromosome aberrations test in the future (Marzin, 1997; Miller et al., 1997; Kirkland, 1998).
The assay is based on addition of the actin inhibitor cytochalasin B (cytB) to the culture medium, which inhibits cell division but not nuclear division (Carter, 1967), resulting in the induction of binucleate cells. Consequently, scoring of micronuclei and/or aneuploidy can be restricted to those cells that have undergone a nuclear division (binucleate cells) in the presence of the test compound (Fenech, 1997). Furthermore, by staining the centromeres by means of FISH with a centromeric probe, this assay can discriminate between aneugenic and clastogenic events. The presence of a centromere in the MN indicates the presence of a whole chromosome and is considered as chromosome loss, whereas the absence of a centromere in the MN is indicative of chromosome breaks (Russo et al., 1996; de Stoppelaar et al., 1997; Schuler et al., 1997). Another advantage of the assay is that non-disjunction and chromosome loss, the two basic mechanisms by which aneuploidy arises, can be determined simultaneously in the same binucleate cells by applying FISH with chromosome-specific probes (Zijno et al., 1994; Marshall et al., 1996). This is especially valuable since non-disjunction is believed to be a more important and earlier event in the induction of aneuploidy than chromosome loss (Zijno et al., 1996b; Elhajouji et al., 1997). These advantages of the cytokinesis blocked micronucleus assay prompted us to study ways in which this methodology can be applied in vivo.
For this purpose we have used the granuloma pouch assay (GPA) in the rat as a model system. In this assay a pouch is induced by s.c. injection of sterile air under the skin of the back of the rats, resulting in a transient proliferation of the underlying fibroblasts (Maier, 1984; Mohn et al., 1989). After injection of a given test compound into the pouch, the fibroblasts can be isolated and different genotoxic end-points studied.
The present paper describes the application of the cytokinesis block micronucleus assay in the GPA in three different ways: (i) in vivo exposure to carbendazim (CARB) and in vivo induction of binucleate cells by injecting cytB and CARB into the pouch simultaneously; (ii) in vivo exposure to CARB and in vitro induction of binucleate cells by culturing the cells in the presence of cytB; (iii) in vitro exposure to CARB and in vitro induction of binucleate cells by culturing the cells in the presence of CARB and cytB. CARB was used as the test compound because it is a potent aneugen in vitro (Elhajouji et al., 1995, 1997; Marshall et al., 1996) and induces micronuclei in mouse bone marrow (Sarrif et al., 1994). Since CARB has been shown to induce polyploidy in rat sperm (de Stoppelaar et al., 1999), the frequency of polyploidy was also determined in the present assays. Because in the cytokinesis blocked micronucleus assay the frequency of polyploid binucleate and mononucleate cells was decreased after exposure to CARB, which was an unexpected finding, an additional polyploidy assay in vitro was carried out without blocking cytokinesis by cytB.
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Materials and methods |
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Pouch induction and isolation of fibroblasts
Pouches were induced in male Wistar rats [WU(CPB), weighing 200–220 g] as described by Maier (1984) and Mohn et al. (1989). Twenty-five millilitres of sterile air were injected into the scapular area on the backs of the rats, resulting in an air pouch, which will remain for ~10 days (Figure 1A
). Forty-eight hours thereafter, CARB (Aldrich) or tricaprylin (Fluka), with or without cytB (Sigma), was injected into the pouch.
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Three or four days after pouch induction the fibroblasts were isolated as follows. Rats were killed by O2/CO2 asphyxiation and the backs of the rats were shaved and disinfected. Granuloma pouch tissue was removed from the skin (Figure 1B and C
) and kept in 1 ml of phosphate-buffered saline (pH 7.4, 4°C). The pouch tissue was dissected into small pieces and subsequently treated three times with a 0.25% trypsin, 0.02% EDTA solution by gentle stirring for 20 min at 37°C. After each incubation, released cells were collected, resuspended in 10 ml culture medium (Dulbecco's modified essential medium supplemented with 10% fetal calf serum, glutamine, gentamycin and fungizone) and stored at 4°C. The cells were filtered through an `open chamber filter' (NPBI, Emmer-Compascuum, the Netherlands) and centrifuged for 10 min at 1000 r.p.m. Cell pellets were resuspended and the number of cells determined (Coulter Counter). For determination of viability, 300 cells of each suspension were seeded in a Petri dish with culture medium. After 7–8 days of incubation, the clones were stained, counted and the cloning efficiency was determined.
Cytokinesis blocked micronucleus assays
In vivo treatment with CARB/in vivo induction of binucleate cells (in vivo/in vivo assay).
For determination of the maximum percentage of binucleate cells that could be recovered from the pouch, different amounts of cytB were injected into the pouch (0–2 mg/pouch in 1 ml tricaprylin). Two or three rats were used per dose group; negative controls were untreated or injected with 1 ml tricaprylin. Forty-eight hours later the fibroblasts were isolated and cultured overnight to remove the dead cells, blood cells and debris. The following day the cells were collected and slides were prepared.
In the cytokinesis blocked micronucleus assay, various amounts of CARB together with cytB (1 mg) were dissolved in 1 ml tricaprylin and injected into the pouch (Figure 2A). In the first experiment rats were treated with 0, 10, 25 or 50 mg CARB/pouch (groups of four, six, one and five rats, respectively) and in the second experiment with 0, 2.5, 5 or 10 mg CARB/pouch (6 rats/group). After 48 h rats were sacrificed and the fibroblasts were isolated. In the first experiment cells from all rats of the same group were pooled and in the second experiment cells from 3 rats/dose group were pooled. The cells were cultured overnight and collected the following day to prepare micronucleus slides.
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In vivo treatment with CARB/in vitro induction of binucleate cells (in vivo/in vitro assay). CARB (0, 10 or 50 mg in the first experiment and 0, 2.5, 5 or 10 mg in the second experiment) in 1 ml tricaprilyn was injected into the pouches of six rats (Figure 2B
). Twenty-four hours later rats were killed and the fibroblasts were isolated. Cells from six (first experiment) or three (second experiment) rats were pooled and cultured for 72 h in the presence of cytB (6 µg/ml). Cells were harvested and micronucleus slides were prepared.
In vitro treatment with CARB/in vitro induction of binucleate cells (in vitro/in vitro assay).
The primary fibroblasts used for the in vitro assays were obtained from male RIV:Tox Wistar rats (RIVM, weighing 200–220 g). Pouches were induced in 10 rats and 3 days later the fibroblasts were isolated, cultured and frozen in liquid nitrogen at passage 4. Before freezing, the cells from all rats were pooled to make one batch, which was used for the in vitro assays (Figure 2C1). Twenty-four hours after seeding 1x106 cells into culture flasks (175 cm2), the culture medium was replaced with medium containing 6 µg/ml cytB and 0, 0.5, 1, 2.5 or 5 µg CARB/ml culture medium. CARB was dissolved in dimethylsulphoxide (DMSO), which was added to the culture medium at a final concentration of 100 µl/10 ml culture medium. Thirty hours after the start of treatment the cells were harvested and micronucleus slides were prepared.
In vitro treatment with CARB without cytB (polyploidy assay in vitro).
Primary fibroblasts from the same batch as in the in vitro cytokinesis blocked micronucleus assay were used (Figure 2C2). Twenty-four hours after seeding 0.5x106 cells into culture flasks (80 cm2), the medium was replaced with medium containing 0, 1, 2.5, 5, 10 or 20 µg CARB/ml medium. CARB was dissolved in DMSO, which was added to the culture medium at a final concentration of 100 µl/10 ml culture. Thirty and 48 h after the start of treatment the cells were harvested and slides prepared following standard procedures.
Preparation of micronucleus slides.
Cells were harvested and centrifuged for 8 min at 1000 r.p.m. The pellet was resuspended in cold 0.075 M KCl followed by immediate centrifugation. Cells were fixed in methanol:acetic acid (3:1) and dropped onto slides. Cells from the in vitro/in vitro assay were fixed twice in methanol:acetic acid before they were dropped onto slides. Slides were stored at –20°C until use. Before hybridization slides were `baked' on a hotplate at 56°C for 2–4 h.
Fluorescence in situ hybridization
The different probe combinations used in the different assays are shown in Table I. Probes were labelled by nick translation (2 h at 16°C) with biotin-dUTP or with digoxigenin-dUTP (in the case of dual colour FISH), both purchased from Boehringer Mannheim. Labelled probe was kept at –20°C in nick translation buffer until use.
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For each FISH the probe mixture was freshly prepared (final solution 20 ng of each probe and 10 µg herring sperm DNA in 55% formamide, 10% dextran sulphate, 2x SSC) which was denatured at 80°C for 10 min. The slides were denatured at 70°C for 3–4 min in 70% formamide, 2x SSC, pH 7.0, and dehydrated in an ethanol series. Following application of 10–15 µl of probe mixture, hybridization was performed at 37°C overnight in a humidified chamber. Two series of post-hybridization washings were carried out, one in 2x SSC at 65°C (3x10 min) and one in 0.1x SSC at 45°C (3x10 min). In the case of hybridization with the chromosome Y probe the second washing was carried out at 65°C.
Visualization of the probes was achieved by a series of incubations in blocking buffer (4x SSC, 0.5% Boehringer Blocking Reagent, 0.02% NaN3) containing the detection reagents. In each series of slides from the same group, half of the slides were incubated with avidin–FITC (Vector), biotinylated-anti-avidin (Vector) + anti-digoxigenin–rhodamine (Boehringer Mannheim) and avidin–FITC + anti-sheep-lissamin–rhodamine (Jackson Immunoresearch). The other half of the slides was incubated with avidin–cy3 (Jackson Immunoresearch), biotinylated-anti-avidin + anti-digoxigenin–FITC (Boehringer Mannheim) and avidin–cy3. After dehydration in ethanol the slides were embedded in anti-fade medium containing propidium iodide/acridine orange (Sigma), in the case of single colour FISH, or 4,6-diamidino-2-phenylindole (DAPI; Sigma), in the case of dual colour FISH, as counterstain.
Scoring of the slides
Scoring criteria were used as described elsewhere (de Stoppelaar et al., 1997). In the in vitro assays at least 1000 binucleate cells were scored. Due to the low frequency of binucleate cells on the slides of the in vivo assays it was decided to score as many binucleate cells as possible on two or three slides instead of the usual 1000 cells. A result was considered significantly different from the control group if the 95% confidence intervals did not overlap each other.
On the micronucleus slides hybridized with the rat satellite I probe the fraction of binucleate cells, the frequency of binucleate cells with one or more micronuclei and the presence of a hybridization signal in the micronuclei were determined (Table I). In addition, in the in vivo/in vivo assay and the in vitro/in vitro assay at least 2000 mononucleate cells were scored for micronuclei and chromosome loss.
After hybridization with chromosome-specific probes on micronucleus slides, the distribution of the hybridization signals among the two daughter nuclei (and micronuclei, if present) of the binucleate cells was determined. The evaluation of non-disjunction and chromosome loss was restricted to those cells containing four hybridization signals of the respective probes (Table I). In addition, binucleate and mononucleate cells (at least 1000) were scored for the occurrence of polyploidy. In binucleate cells polyploidy was defined as cells having at least eight hybridization signals per probe or at least eight signals of one probe and at least seven of the other probe, the latter being likely to represent polyploid cells with overlapping signals. Mononucleate cells should contain at least four signals of one probe and at least three signals of the other probe in order to be scored as polyploid.
In the in vitro assay without cytB, polyploidy was scored by determining the number of hybridization signals from the two probes in at least 1000 nuclei (Table I). The criteria for scoring of polyploidy were the same as in the in vitro micronucleus assay. Two different slides per culture were scored, for at least 500 cells/slide.
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Results |
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In vivo/in vivo assay
Determination of cytB dose. To determine the amount of cytB optimal for the induction of binucleate cells, a range-finding experiment was carried out in which different amounts of cytB were injected into the pouch. In previous experiments in the GPA, the cell cycle of untreated fibroblasts was observed to be 18–24 h and that of treated fibroblasts up to 48 h. Consequently, in order to ensure that the proliferating cells had divided in the presence of cytB, the fibroblasts were isolated 48 h after injection of cytB into the pouch. The percentage of binucleate cells increased from 0.5% in control rats to an average of 4% at a dose of 1 mg cytB (with values up to 5–6% in individual rats; Figure 3
). Since higher amounts of cytB did not increase the frequency of binucleate cells, 1 mg cytB/pouch was used in the in vivo/in vivo experiments.
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Slides hybridized with the general centromeric probe. In the first in vivo/in vivo assay (Figure 2A
) a very significant induction of micronuclei was observed, which was apparent even at the lowest dose tested (10 mg CARB/pouch; Table II). CARB treatment had no effect on the percentage of binucleate cells. Chromosome loss, which was determined by scoring the micronuclei for the presence of a hybridization signal of the general centromeric probe 18-5 (Figure 4A), was increased in the treated groups. Expressing chromosome loss as the number of signals in micronuclei per 1000 binucleate cells, chromosome loss in control rats was 13.2 whereas in treated rats it was increased to 160–220 (Table III). Since the lowest dose of CARB tested induced a major increase in chromosome loss, the experiment was repeated using 10 mg/pouch as the highest dose.
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In the second experiment a decrease in the percentage of binucleate cells was observed, from an average of 3.8% in controls to an average of 1.5% in the highest dose group (Table II
). The micronucleus frequency in binucleate cells was significantly and dose-relatedly increased in the treated groups, from 1.2% in the controls to an average of 12% in the highest dose group (Table II). Chromosome loss was increased significantly after CARB treatment, from an average of 8/1000 cells in controls to 130–170/1000 cells in the highest dose group (Table III).
In order to determine if cells that are exposed to CARB can escape the cytokinesis block, micronuclei and chromosome loss were also scored in mononucleate cells. The micronucleus frequency in mononucleate cells increased from 0.2% in controls to 0.9% in the highest dose group, whereas the frequency of chromosome loss increased from 1.2 to 6.5 signals in micronuclei per 1000 mononucleate cells (Figure 5).
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Slides hybridized with chromosome-specific probes. On slides hybridized with two probes for chromosome 19 (see Table I
) binucleate cells were scored as normal when each nucleus contained two signals of both probes (Figure 4B). Three signals of each probe in one nucleus and one signal of each probe in the other nucleus was considered a non-disjunction event of chromosome 19 (Figure 4C). Loss of chromosome 19 was scored when a micronucleus contained a hybridization signal of both probes (Figure 4D). Cells with an unequal distribution of the hybridization signals were also observed, for instance a 3–1 distribution of signals from one probe and a 2–2 distribution of the other probe or cells with a hybridization signal of only one probe in the micronucleus. This is probably due to a break in the chromosome (and perhaps a subsequent translocation), enabling the fluorescent signals to segregate independently of each other. In the remaining part of the manuscript these events will be referred to as `breaks'.
Non-disjunction frequencies for chromosome 19 increased with dose after treatment with CARB, from an average of 6 non-disjunction events/1000 binucleate cells in control rats to an average of ~65/1000 cells in the highest dose group (Figure 6A). Chromosome loss was practically absent in controls, whereas in the highest dose group an average of 32 was scored per 1000 cells. Cells with chromosome 19 breaks also increased in the treated groups, but without an obvious relation to the dose. The total frequency of aberrant cells was ~100 aberrations/1000 binucleate cells in the highest dose group, compared with an average of 12/1000 BC in the control group (Figure 6A).
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In order to confirm these high frequencies of malsegregation, FISH with probes for chromosome 4 and Y (see Table I
) was performed on slides from control rats and rats treated with the highest dose of CARB (10 mg/pouch). Scoring of at least 2 slides/group for chromosome loss and non-disjunction again showed a major increase in chromosome loss and non-disjunction after treatment with CARB, which confirmed the high aneuploidy frequencies detected with chromosome 19 (Table IV).
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Polyploidy. Binucleate as well as mononucleate cells from slides hybridized with the chromosome-specific probes were also scored for polyploidy (Table I
; see for example Figure 4E). Induction of polyploidy was not observed in binucleate or in mononucleate cells hybridized with the chromosome 19 probes (Figure 7A) or with the chromosome 4 and Y probes (data not shown). On the contrary, the frequencies of polyploid binucleate and mononucleate cells were decreased in the treated cultures with all probes scored.
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In vivo/in vitro assay
After injecting CARB into the pouch and culturing the isolated fibroblasts in vitro in the presence of cytB (Figure 2B
), the total number of viable cells recovered from the pouch was significantly decreased (data not shown). In the two highest dose groups only 19–120 binucleate cells could be scored on two slides (data not shown), which consequently resulted in a very low number of micronuclei scored for the presence of hybridization signals. It was therefore decided not to evaluate this experiment any further.
In vitro/in vitro assay
Slides hybridized with the general centromeric probe.
In the in vitro cytokinesis blocked micronucleus assay (Figure 2C1) the percentage of binucleate cells was decreased at the highest dose and the frequency of binucleate cells with more than one micronucleus was increased with dose (Table V). In the highest dose group, 35% of the binucleate cells contained one or more micronuclei, most of which contained one or more hybridization signals of the general centromeric probe 18-5. Thus, a very pronounced increase in chromosome loss was observed after CARB treatment, up to 487/1000 cells (Table VI). Due to the many micronuclei and the toxicity (low percentage of binucleate cells) at 5 µg/ml, only the dose range 0–2.5 µg/ml was scored for the other evaluations.
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In mononucleate cells, the micronucleus frequency was 1.5% in the control culture and increased up to 5% in the culture treated with 2.5 µg/ml (data not shown). Chromosome loss was increased from 3/1000 mononucleate cells in the control culture to 32/1000 mononucleate cells in the 2.5 µg/ml culture (data not shown). Thus, as in the in vivo/in vivo assay, the induction of chromosome loss in mononucleate cells was much lower than in binucleate cells.
Slides hybridized with the chromosome 19 specific probes.
Similar to the analysis of the in vivo/in vivo assay non-disjunction, chromosome loss and chromosome breaks were scored using two probes for chromosome 19. Non-disjunction and chromosome loss were not observed in control cultures (Figure 6B). In the cultures treated with 1 and 2.5 µg/ml CARB chromosome loss was significantly increased to a maximum of 40/1000 binucleate cells and non-disjunction events increased to 75/1000 binucleate cells at the highest dose. Chromosome breaks were increased in the culture treated with 2.5 µg/ml CARB, but not significantly (Figure 6B).
Polyploidy.
Slides hybridized with the chromosome 19 probes were also scored for the occurrence of polyploidy in mononucleate and binucleate cells. In the binucleate cells the frequency of polyploidy was 4.5% in control cultures and decreased significantly to 0.3% in the 2.5 µg/ml CARB culture (Figure 7B). In the mononucleate cells the frequency of polyploid cells decreased significantly from 10% in the control culture to 5% in the 2.5 µg/ml CARB culture (Figure 7B).
In vitro assay without cytB
The absence of polyploidy induction by CARB in the cytokinesis blocked micronucleus assay triggered the question whether cytB may interfere with polyploidy induction. In order to test this idea, an additional in vitro assay was carried out without using cytB (Figure 2C2), with concentrations ranging from 0 to 20 µg/ml CARB. After dual colour FISH with probes specific for chromosome 4 and for chromosome 19, the slides were scored for polyploidy (see for example Figure 4F). Results showed a significant induction of polyploid cells in the treated cultures from 2.5 µg/ml and above (Figure 7B), at both 30 and 48 h after initiation of treatment. The frequency of polyploid cells increased from ~5% in the control cultures to 40–50% at 20 µg/ml.
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Discussion |
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Induction of binucleate cells in vivo
For application of the cytokinesis blocked micronucleus assay in vivo a technical difficulty is to ensure that a sufficient quantity of cytB reaches the target cells in order to block cytokinesis effectively. Unless a significant local exposure of the target cells can be achieved, large amounts of cytB have to be injected into the animal. From a toxicological as well as an economic point of view this is not desirable. The GPA offers a unique opportunity to achieve a high local exposure of target cells to cytB, without a concomitant high systemic exposure. In this assay proliferation of fibroblasts is induced by mechanical stress caused by pouch induction, and when proliferation is at its maximum (48 h after pouch induction; Maier, 1984
) cytB (with or without the test compound) can be injected into the pouch (Figure 2A). In this way a direct exposure of the proliferating cells to cytB was achieved and binucleate cells were induced in vivo (Figure 3).
There has been one recent report concerning the induction of binucleate cells in vivo, namely in mice carrying different kinds of transplanted tumours (Devi et al., 1998). Three i.p. injections of cytB (3 + 2 + 2 mg/kg) at 12 h intervals resulted in a maximum percentage of binucleate tumour cells of 7.5%. Since these results were obtained in transplanted tumour cells they are not comparable with the present results, which were obtained in an animal's own somatic cells. Injection of up to 2 mg cytB into the pouch resulted in a maximum recovery of 4–6% binucleate cells in individual rats (Figure 3). Injecting higher amounts of cytB was not considered since the maximum yield of binucleate cells was already achieved at 1 mg cytB/pouch. The limiting factor in the induction of binucleate cells in the present assay is probably the number of fibroblasts exposed to cytB (and CARB). CytB (and CARB) is insoluble in water and is therefore applied as a suspension in tricaprylin. Due to the watery environment in the pouch, cytB (and CARB) will either precipitate in the pouch or remain confined to the oil phase and cluster on the bottom of the pouch. However, the isolated fibroblasts are mostly recovered from the `upper' part of the pouch (see Figure 1B).
Despite the apparent low yield of binucleate cells, it is possible to obtain enough binucleate cells from the pouch tissue for analysis of micronuclei and aneuploidy. The total yield of viable cells from one pouch varied between 0.3x106 and 1.9x106 cells in the cytB-treated animals and between 0.2x106 and 0.8x106 in the CARB + cytB-treated animals (data not shown). Taking the lowest yield of cells and the lowest percentage of binucleate cells observed (1%; see Table II), the total number of binucleate cells recovered from the pouch is still calculated to be 20 000. In order to ensure that after culturing and slide preparation there were still enough binucleate cells available for analysis of aneuploidy with different probe combinations, the cells from three rats were pooled.
An alternative approach for the induction of binucleate cells, namely in vitro culturing of the fibroblasts in the presence of cytB after they had been exposed to CARB in vivo, was not successful due to the low viability of the cells. Since it is essential that the cells undergo their first cell division in vitro in the presence of cytB, the fibroblasts were recovered 24 h after exposure instead of 48 h, as in the in vivo/in vivo assay. This shorter time interval may be insufficient for the cells to recover from the insult of injection of the test compound. The subsequent isolation of the fibroblasts (with multiple trypsin treatments) and the presence of cytB in the culture medium will hamper recovery of the cells even more, resulting in a low viability of the cells.
High aneuploidy frequencies induced by CARB
The present results show that CARB is a very potent aneugen, irrespective of which probe was used for detection by FISH. This is best illustrated by the results obtained with the chromosome-specific probes, since both non-disjunction and chromosome loss are measured with these probes. In the in vivo/in vivo assay the average total malsegregation frequency (chromosome loss + non-disjunction) of chromosomes 4, Y and 19 in the high dose groups together was 290 events/1000 binucleate cells. Assuming that malsegregation occurs randomly among the chromosomes, the equivalent for the whole genome (x42/5) is 2436 malsegregated chromosomes/1000 binucleate cells, meaning that each cell must contain two or three malsegregated chromosomes on average. This high frequency of aneuploidy is not unexpected. CARB and cytB are applied in the same solution, which is not miscible with the watery environment in the pouch. Consequently, the binucleate cells represent a highly selected population of exposed cells that have undergone a nuclear division in the presence of CARB. Furthermore, CARB is a direct acting agent so that the proliferating pouch cells exposed to CARB are likely to become aneuploid.
In the in vitro cytokinesis blocked assay in the same target cells, similarly high aneuploidy frequencies of chromosome 19 were observed in the 2.5 µg/ml culture. Again calculating the equivalent for the whole genome, total malsegregation frequency would be 2460 chromosomes/1000 binucleate cells, comparable with the results obtained in the in vivo assay. High frequencies of CARB-induced aneuploidy in vitro have been observed previously in cytokinesis blocked human lymphocytes (Marshall et al., 1996; Elhajouji et al., 1997). Marshall et al. (1996) measured non-disjunction and chromosome loss in binucleate human lymphocytes with six chromosome-specific probes and observed CARB-induced aneuploidy frequencies as high as in the present assay in a similar dose range.
Comparison of chromosome loss and non-disjunction measured with the different probes
In the experiments by Marshall et al. (1996) non-disjunction frequencies induced by CARB were about twice the chromosome loss frequencies. The present results are in agreement with this observation (Figure 6A and B and Table IV) and support observations made by others that non-disjunction is the prevalent error leading to aneuploidy in cytokinesis blocked human lymphocytes (Marshall et al., 1996; Zijno et al., 1996b; Elhajouji et al., 1997).
Comparison of chromosome loss or non-disjunction frequencies of the different chromosomes did not reveal any significant differences. However, although not significantly, the chromosome loss detected with the chromosome 19 probes was clearly higher than with the other probes (including the general centromeric probe when corrected for the number of chromosomes detected by this probe; data not shown). This was observed in both the in vivo/in vivo assay and the in vitro/in vitro assay. In an in vitro micronucleus test with diethylstilbestrol (DES) in which a few hundred micronuclei were scored for the presence of hybridization signals (de Stoppelaar et al., this issue), a similar observation was made. Although the observed differences in chromosome loss were not statistically significant, it is a recurring observation and may therefore be more than coincidental.
Preferential loss of smaller chromosomes has been observed before in human lymphocytes (see for example Richard et al., 1993), but due to the method of slide preparation it cannot be ruled out that these losses are artefactual. More comparable with the present analysis are the results of Wuttke et al. (1997) and Caria et al. (1996), who observed an over-representation of acrocentric chromosomes (which in humans are generally smaller than metacentric chromosomes) in colchicine-induced micronuclei of human cells in vitro. In contrast, in spontaneous micronuclei this over-representation was not observed (Scarpato et al., 1996). Further studies are needed in order to draw definite conclusions on this point.
Using two probes on the same chromosome
For the determination of non-disjunction of chromosome 19 two probes were used simultaneously, hybridizing on either side of the centromere. Using two probes for the same chromosome instead of a single one allows distinction between actual aneuploidy events and other events such as chromosome breaks. This improves the sensitivity of the assay, illustrated previously in an in vitro cytokinesis blocked micronucleus assay in pouch fibroblasts with DES (de Stoppelaar et al., this issue). These results showed that as well as non-disjunction, DES also induced a small increase in chromosome breaks, which was confirmed by the results obtained with the general centromeric probe (de Stoppelaar et al., 1997). In the present analysis, a small increase in chromosome 19 breaks was observed in the treated groups in both the in vivo/in vivo and the in vitro/in vitro assays, but the increase was not statistically significant and was without an obvious dose–response relationship. This confirms the predominantly aneugenic action of CARB and the absence of a clear clastogenic action.
Aneuploidy in mononucleate cells
Elhajouji et al. (1998) observed an induction of micronuclei in mononucleate cells in an in vitro cytokinesis blocked micronucleus assay by CARB and other aneugens (colchicine, nocodazole and mebendazole). With the two clastogenic agents mitomycin C and methyl methane sulphonate induction of micronuclei in mononucleate cells was not observed in the same assay. The finding that aneugens can induce micronuclei in mononucleate cells in vitro was confirmed in the present in vitro/in vitro assay. The induction of micronuclei and chromosome loss by CARB in mononucleate cells was, however, much smaller than in binucleate cells, which is in agreement with the observations of Elhajouji et al. (1998). In the in vivo/in vivo assay a similar result was obtained, induction of chromosome loss in mononucleate cells being much lower than in binucleate cells. This finding illustrates that the binucleate cells represent a selected population of CARB-exposed cells (as discussed previously). Thus, for determination of whether or not a certain compound induces aneuploidy in the cytokinesis block assay, scoring of only binucleate cells is sufficient.
Polyploidy
The finding that CARB induced polyploidy in rat sperm (de Stoppelaar et al., 1999) prompted us to score for polyploidy in the cytokinesis blocked micronucleus assays described here. In both the in vivo/in vivo and the in vitro/in vitro assays induction of polyploidy was not observed, neither in mononucleate nor binucleate cells. On the contrary, the frequency of polyploidy was decreased after treatment with CARB (see Figure 7), which was rather unexpected since most aneugens also induce polyploidy (for a review see European Centre for Ecotoxicology and Toxicology of Chemicals, 1997). This finding is in contrast to the results observed by Minissi et al. (1999) and Zijno et al. (1996a), who observed higher frequencies of polyploid nuclei induced by colchicine and vinblastin in cultures treated with cytB, compared with cultures untreated with cytB.
To investigate induction of polyploidy without the possible interference of cytB, we performed an additional in vitro assay with CARB, omitting the incubation with cytB. A dose-related induction of polyploidy was observed, which was apparent at 2.5 µg/ml and increased at higher concentrations. It appears that polyploidy induction occurs at higher doses of the test compound than aneuploidy induction, which was very prominent at 2.5 µg/ml. This dose effect has been observed by others as well (Minissi et al., 1999) and is not an unexpected finding, considering that for polyploidy induction the spindle and microtubules need to be more severely disturbed than for induction of aneuploidy. However, the dose effect cannot explain why the polyploidy frequency decreased after CARB treatment in the present assays with cytB. It is likely that cytB interfered with the induction of polyploidy, although in a different way than observed by other investigators (Zijno et al., 1996a; Minissi et al., 1999). Thus, the cytokinesis blocked micronucleus assay is not a suitable assay to obtain information on the polyploidy-inducing properties of the test compound.
Usefulness of the in vivo cytokinesis blocked micronucleus assay in the GPA
This is to our knowledge the first report of an in vivo cytokinesis blocked micronucleus assay in somatic cells. Although the percentage of binucleate cells recovered from the pouch was between 1 and 5%, the results were quite reproducible. At 10 mg CARB/pouch, the frequencies of micronuclei and chromosome loss were similar in two independent assays (Tables II and III). The only difference between the two assays lies in the percentage of binucleate cells recovered from the pouch, but this difference does not seem to influence the results. The fact that CARB is a potent aneugen in vitro in the cytokinesis blocked micronucleus assay was already known (Marshall et al., 1996; Elhajouji et al., 1997). In the present paper the potency of CARB was confirmed in vivo and the induced aneuploidy frequencies were similar to those observed in an in vitro assay in the same cells. Aneuploidy was induced at 2.5 mg/pouch (and above) which, considering that the rats weighed 200–220 g, is equivalent to ~12 mg/kg body wt. In mouse bone marrow micronucleus assays with CARB the lowest positive dose was 500 mg/kg body wt (Pandita, 1988; Barale et al., 1993; Sarrif et al., 1994). The lowest dose tested in these assays was 66 mg/kg body wt, which did not induce micronuclei (Sarrif et al., 1994). In rats an oral dose of CARB of 800 mg/kg body wt did not induce micronuclei in peripheral blood erythrocytes (de Stoppelaar et al., 1999). Thus, the present assay is a more sensitive assay for micronucleus induction by CARB than the (mouse) bone marrow micronucleus assay. Furthermore, the potency of CARB in the present assay was much higher than in the bone marrow micronucleus assay. The higher sensitivity of the GPA is not surprising considering the very direct and local exposure of the target cells and the selection of exposed cells by scoring only the binucleate cells. Another important advantage of the cytokinesis blocked assay in vivo over the bone marrow micronucleus assay is that non-disjunction can be studied in addition to chromosome loss. This is especially important in the light of the fact that non-disjunction seems to be the prevalent error leading to aneuploidy (as discussed previously). In conclusion, for studying the aneugenic effects of spindle poisons like CARB, and possibly also for other aneugenic or clastogenic agents, the GPA in combination with the cytokinesis block micronucleus assay may be a preferable alternative to the bone marrow micronucleus assay.
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We thank Marjo Poelen and Paul Reulen for biotechnical assistance, Ernst Rozendal for preparing the pictures and Prof. Dr A.T. Natarajan for discussions and critically reading this manuscript.
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1 To whom correspondence should be addressed. Tel: +31 30 274 2021/2196; Fax: +31 30 274 4446; Email: J.van.Benthem{at}rivm.nl
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Received on September 7, 1999; accepted on November 18, 1999.
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  J. M. de Stoppelaar, P. Faessen, E. Zwart, L. Hozeman, H. Hodemaekers, G. R. Mohn, and B. Hoebee Isolation of DNA probes specific for rat chromosomal regions 19p, 19q and 4q and their application for the analysis of diethylstilbestrol-induced aneuploidy in binucleated rat fibroblasts Mutagenesis, March 1, 2000; 15(2): 165 - 175. [Abstract] [Full Text] [PDF]
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