Mutagenesis, Vol. 15, No. 1, 17-24,
January 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Trichlorfon induces spindle disturbances in V79 cells and aneuploidy in male mouse germ cells
1 Institute of Mammalian Genetics and 2 Institute of Radiation Biology, GSF-National Research Centre for Environment and Health, Ingolstaedter Landstrasse, D-85764 Neuherberg, Germany and 3 Department of Toxicology, Medical College, Qingdao University, Qingdao 266021, People's Republic of China
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Abstract |
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In order to assess the effects of trichlorfon on cell division and on aneuploidy induction, we conducted an in vitro assay for spindle disturbances using V79 cells and an in vivo assay for aneuploidy induction in meiosis of male mice using multicolour fluorescence in situ hybridization (FISH) with epididymal sperm. In the in vitro assay, the chemical caused a concentration-dependent increase in the incidence of initial and full c-mitoses in the dose range 40–120 µg/ml trichlorfon. The mitotic index (MI) was decreased between 40 and 100 µg/ml trichlorfon, whereas at 120 µg/ml the MI was back to the control level, coinciding with the dramatic increase in c-mitoses. The results confirm that trichlorfon is a potent spindle poison in V79 cells. In the in vivo multicolour FISH assay, administration of trichlorfon to male mice at single doses of 200, 300 and 405 mg/kg caused a dose-dependent increase of the frequencies of disomic sperm (0.068, 0.074 and 0.134%, respectively) compared with the corresponding controls (0.046, 0.042 and 0.056%, respectively). The prevalence of X-X-8 and Y-Y-8 sperm suggests that trichlorfon affected chromosome segregation predominantly during the second meiotic division. Diploid sperm were not induced by trichlorfon treatment, indicating that no meiotic block occurred. It is concluded that trichlorfon is a potent spindle poison in V79 cells and induces aneuploidy in mouse spermatocytes during meiosis.
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Introduction |
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Trichlorfon (dimethyl 2,2,2-trichloro-1-hydroxyethylphosphonate), as an insecticide, has been used on a large scale in crop protection and in the field of hygiene since 1952 (Lorenz et al., 1955
). Under the name metrifonate, trichlorfon made its way into veterinary and human medicine, e.g. for the treatment of schistosomiasis in man (Lebrun and Cerf, 1960), cysticercosis in pig and in man (Malagón, 1989) and the experimental treatment of Alzheimer's disease in man (Hallak and Giacobini, 1989; Tariot et al., 1997). Its widespread use is a potential source of exposure for both workers and the general population.
Trichlorfon use is broadly based on its action as an inhibitor of acetylcholine esterase (Reiner et al., 1975). It is unique in that it has been claimed not to be a direct acting cholinesterase inhibitor but to be transformed non-enzymatically, in water and all biological fluids and tissues at pH values higher than 5.5, into a far more active component, dichlorvos (2,2-dichlorovinyl dimethyl phosphate, DDVP) (Van Die, 1957; Metcalf et al., 1959; Miyamoto, 1959; Nordgren et al., 1978). It has been argued that the biological effects produced by trichlorfon are exerted through the action of this metabolite (Nordgren et al., 1978). Yamano and Morita (1992) also reported that trichlorfon and dichlorvos have been observed to produce increased toxicity in isolated hepatocytes and it has been suggested that the hepatotoxicity observed with trichlorfon is mediated by dichlorvos, which exerts its toxic effects directly or through metabolism to dichloroacetaldehyde. Although this may be primarily the case, at least part of the genotoxic activity is probably due to trichlorfon itself, because it was demonstrated that trichlorfon causes guanine N7 methylation of liver and kidney DNA after in vivo exposure of mice (Dedek, 1981).
Mutagenicity studies with trichlorfon gave positive results in prokaryotes and plants. Trichlorfon was mutagenic in Salmonella typhimurium and Escherichia coli (Poole et al., 1977; Carere et al., 1978; Benigni et al., 1980; Shirasu et al., 1982; Morya et al., 1983; Barrueco et al., 1991) and in yeast (Riccio et al., 1981; Gilot-Delhalle et al., 1983). It also induced chlorophyll mutations (Panda and Sharma, 1980) and chromosomal damage in plants (Logvinenko and Morgun, 1978; de Kergommeaux et al., 1983).
Studies with mammalian cells in vitro also gave positive results. Trichlorfon induced unscheduled DNA synthesis (UDS) in human epithelial cells (Aquilina et al., 1984) and in human fibroblasts (Waters et al., 1982). It induced sister chromatid exchanges (SCE) in Chinese hamster ovary (CHO) cells (Chen et al., 1981; Waters et al., 1982) and in human lymphocytes (Madrigal-Bujaidar et al., 1993). Chromosomal aberrations were observed in CHO cells (Sasaki et al., 1980; Ishidate et al., 1981) and in human lymphocytes (Kurinniy and Pilinskaya, 1977) after trichlorfon treatment. It also induced micronuclei and multinucleated cells and decreased binucleated cells in human lymphoblastoid cell lines (Doherty et al. 1996). Trichlorfon was also reported to cause oncogenic transformation in C3H1OT1/2 CL8 cells (Waters et al., 1981, 1982) and forward mutations in mouse lymphoma L5178Y cells in the absence of metabolic activation (McGregor et al., 1988).
In vivo studies have given contradictory results. Trichlorfon was reported to induce chromosomal aberrations (Kurinniy, 1975; Kuzmenko et al., 1980; Ryazanova and Gafurova, 1980; Nehéz et al., 1987) and SCE (Madrigal-Bujaidar et al., 1993) in mouse bone marrow cells. However, cytogenetic studies after chronic exposure to trichlorfon in drinking water showed no increase in chromosome aberrations in bone marrow cells of mice (Degraeve et al., 1984). Micronuclei were not induced by trichlorfon in mouse bone marrow erythrocytes (Paik and Lee, 1977; Herbold, 1979a; Waters et al., 1982). In humans, cytogenetic studies on lymphocytes from persons following acute intoxication or those occupationally exposed to trichlorfon did show significant increases in the frequencies of chromosome aberrations (Bao et al., 1974; Czeizel, 1994).
Similarly conflicting data were reported in germ cell tests. In the dominant lethal test with mice Dedek et al. (1975) found a significant increase in post-implantation losses at a single dose of 405 mg/kg trichlorfon given i.p. This result, however, could not be confirmed by Becker and Schöneich (1980). Negative results were also reported after a single oral dose of 250 mg/kg trichlorfon (Herbold, 1979b) and after a single i.p. injection of trichlorfon at 100 mg/kg (Degraeve et al., 1984). In addition, a study on mice indicated that trichlorfon did not induce any chromosome damage in germ cells after treatment with 0.5 mg/l in drinking water continuously for 7 weeks (Degraeve et al., 1984). However, in another study on people who had been treated with trichlorfon for a few years to control various intestinal and body parasites, an indication was given that spermatogenesis and sperm mobility might be affected (Wegner, 1970).
Furthermore, an adverse effect of trichlorfon on gonads was demonstrated following oral exposure of mice and rats to 30 and 400 mg/kg body wt in the diet, respectively (World Health Organization, 1991). Trichlorfon was also found to cross the placenta of guinea pigs (Berge and Nafstad, 1986) and teratogenetic effects were reported in hamsters, mice and rats (Staples and Goulding, 1979; Courtney et al., 1986).
Aneuploidy has long been recognized to be causally related to tumorigenesis (Boveri, 1914) and has come to prominence in the past two decades due to its close association with embryonic loss, developmental abnormalities, hereditary disorders and human malignancy (Galloway, 1994; Rew, 1994). In recent years, more attention has been paid to the aneugenic activity of trichlorfon, especially after Czeizel et al. (1993) reported an extreme cluster of congenital abnormalities in a small Hungarian village. A case–control study found an association between trichlorfon exposure and an increase in Down's syndrome cases and, perhaps, some other congenital abnormalities, as well as twins. Due to these observations, it has become increasingly important to understand the effects of trichlorfon on cell division.
As early as 1981, Chen et al. (1981) reported that trichlorfon induced cell cycle delay in V79 cells and pointed out that a delay in cell cycle progression might represent another level of biological damage incurred from treatment with trichlorfon. Likewise, Madrigal-Bujaidar et al. (1993) observed a significant decrease in the replication index (RI) in human lymphocyte cultures exposed to trichlorfon. Doherty et al. (1996), studying the aneugenic activity of trichlorfon in lymphoblastoid cell lines, found that trichlorfon exposure at pH 5 resulted in the induction of both chromosome loss and chromosome non-disjunction. Aneuploidy was also found to be the most prominent form of chromosomal damage observed in peripheral blood lymphocytes of trichlorfon-intoxicated patients (Bao et al., 1974). In vitro studies of mouse oocytes have shown that trichlorfon affects meiotic metaphase I (MMI), producing spindle aberrations and misaligned chromosomes (Yin et al., 1998). Trichlorfon-treated MMI oocytes were not meiotically arrested but progressed to meiotic metaphase II (MMII). The MMII spindles were elongated and disordered with unattached or dispersed chromosomes.
We explored the extent of spindle disturbance exerted by trichlorfon in V79 cells to confirm its in vitro aneugenic potential. Since aneuploidy induction by trichlorfon has not been studied in vivo in mammalian germ cells, we also performed experiments using the multicolour fluorescence in situ hybridization (FISH) method with chromosome-specific DNA probes to identify gain and loss of individual chromosomes in mouse epididymal sperm following trichlorfon treatment of spermatocytes during meiosis.
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Materials and methods |
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Chemicals
Trichlorfon was obtained from Sigma-Aldrich (Steinheim, Germany) and dissolved in double distilled water at pH 5.5 directly before use.
In vitro assay for spindle disturbances
The cell preparation and culture technique have been reported in detail earlier (Adler et al., 1993) and only a brief description is given here. V79 Chinese hamster cells were cultured in Dulbecco's modification of Eagle's minimum essential medium (Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum (FCS) (Flow, Meckenheim, Germany) and antibiotics. Slide cultures (2x105 V79 cells/slide) were set up in Quadriperm dishes (Heraeus, Hanau, Germany) and incubated at 37°C in a humidified atmosphere containing 7% CO2 and 93% air. After 24 h cells were fed with medium containing trichlorfon dissolved in dimethylsulphoxide (DMSO) (Sigma, München, Germany) for 6 h at concentrations ranging from 40 to 120 µg/ml. After exposure, cells were fixed by a mixture of 99% ethanol and glacial acetic acid (3:1). The fixation procedure was repeated three times over a period of 15 min. After air drying, the cells were stained with 2% acetic orcein (Gurr, High Wycombe, UK). In each of two replicates for each concentration of trichlorfon and for the controls with and without DMSO, 500 cells were analysed for spindle disturbances. The mitotic index was determined by scoring 3x1000 cells in different regions of the slides. The polyploidy index gives the number of polyploid mitoses as the percentage of total mitoses analysed.
Animals and treatment
The experiment was performed with male (102/ElxC3H/E1) F1 mice aged 10–14 weeks and weighing 25–29 g. Animals were bred in the GSF mouse colony and were maintained on a 12 h light/dark cycle with mouse pellet food and water ad libitum. The mice were treated with 200, 300 and 405 mg/kg trichlorfon once by i.p. injection. The dose of 405 mg/kg was selected on the basis of positive dominant lethal results reported in the literature (Dedek et al., 1975). Groups of five males were randomly assigned to treatment and concurrent solvent control groups. Concurrent controls were prepared with every dose group in order to code the slides and avoid scoring biases. The injected volume was 0.1 ml/10 g body weight. Mice were killed 22 days after the treatment and sperm were collected from the cauda epididymis.
The multicolour FISH protocol included preparation of slides, isolation of epididymal sperm, slide preparation, production of DNA probes, decondensation of epididymal sperm, denaturation of target DNA, in situ hybridization, washings and detection of hybridization signals. All of these methods were described earlier (Adler et al., 1996; Lowe et al., 1996; Schmid et al., 1999) and were used with some modifications.
Slide preparation from sperm
Epididymal sperm were isolated using the method of Lowe et al. (1996). Both epididymes were removed surgically, the cauda epididymes were separated, placed in a Petri dish and several partial incisions were made with iris scissors. Then, both caudae epididymes from one male were put into an Eppendorf cup filled with 300 µl of FCS. The cups were placed on an Eppendorf incubator at 32°C for 30 min to allow the sperm to actively leave the organs. Subsequently, the organs were removed from the cups and the sperm suspensions were stored at –20°C. Thawed sperm suspensions (7 µl) were pipetted onto ethanol-cleaned dry glass slides. Unfixed cells were smeared across the slide and the slides were dried by first placing them in an Eppendorf incubator at 80°C for 5 min and then by drying at room temperature for 2 days. The slides were coded and stored at –20°C under nitrogen until use.
Prior to in situ hybridization, the slides were taken out and placed at room temperature for 30 min. Thereafter, the sperm were decondensed by putting the slides in a Coplin jar containing 10 mM dithiotreitol (DTT) (Sigma, Deisenhofen, Germany) in 0.1 M Tris–HCl buffer, pH 8.0, for 30 min on ice followed by incubation in 4 mM lithium 3,5-diiodosalicylic acid (LIS) (Sigma) in 0.1 M Tris–HCl buffer, pH 8.0, for 30 min at room temperature. The slides were again air dried completely before they were used for hybridization.
Preparation of DNA probes
Chromosome-specific probes for chromosomes 8 (Boyle and Ward, 1992), X (Disteche et al., 1985) and Y (Bishop and Hatat, 1987) were used for multicolour fluorescence in situ hybridization. Plasmid DNA for chromosomes 8 (clone 84a and 85e), X (clone DXWas70) and Y (clone pY353/B) were transformed in Escherichia coli XL1-blue. DNA isolations were made using the Qiagen Plasmid Maxi Kit (Qiagen, Chatsworth, MD).
The chromosome 8 probe was labelled using the Gibco Nick Translation System with bio-dUTP (Boehringer, Mannheim, Germany). The chromosome X probe was labelled with a combination of bio-16-dUTP and dig-11-dUTP (Boehringer). The chromosome Y probe was labelled using the Gibco Nick Translation System with dig-dUTP (Boehringer).
Three chromosome FISH procedure
Hybridization was performed according to a modified method of Pinkel et al. (1986, 1988). Briefly, labelled probes were mixed with Master Mix 2.1 (55% formamide, 10% dextran in 1x SSC) and denatured at 78°C for 8 min. The sperm on slides were denatured in 70% formamide (in 2x SSC, pH 7.0) at 78°C for 8 min, dehydrated in an alcohol series (70, 90 and 100, 2 min each) and dried on a slide warmer at 37°C for ~3 min before application of the denatured hybridization probe mixture.
Hybridization was carried out for 24–48 h in a moist chamber at 37°C. Post-hybridization washing consisted of two steps: 15 min in 50% formamide (2x SSC, pH 7.0) at 45°C and 30 min in PN buffer at 37°C. The probes were detected with streptavidin-Cy3 (chromosome 8), and anti-dig-FITC (Y chromosome) or a combination of both (X chromosome). Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (20 ng/ml PBS) for 10 min at room temperature and cover-slipped in Vectashield (Vector Laboratories, Burlingame, CA). Slides were stored at 4°C in the dark.
Scoring
Fluorescent images were viewed under a Zeiss Axiopan Fluorescence Photo Microscope (Zeiss, Jena, Germany) equipped with the following filters: triple filter (triple band-pass filter set no. 61000; Chroma Technology, Brattleboro, VT) for simultaneous visualization of green (FITC) fluorescence, yellow (FITC + Cy3) fluorescence, red (Cy3) fluorescence, hybridization domains and the blue (DAPI) fluorescence of the sperm nuclei; individual filters for each of the fluorochromes [FITC, HQ 480/40 and HQ 535/50; Cy3, HQ 535/50 and HQ 610/75 (Chroma Technology); DAPI, BP 365 and LP 397 (Zeiss)] to digitize the image. In this study at least 10 000 sperm/animal were examined microscopically for the number of colour domains they contained. Records of every sperm with an abnormal number of hybridization domains were taken by digitizing the microscopic image with the computer program ISIS3 (MetaSystems, Altlussheim, Germany).
The sperm were assigned to specific fluorochrome phenotypes as determined by the combination of fluorescence signals in each sperm: X-8, Y-8 as normal sperm; X-X-8, Y-Y-8, X-Y-8, X-8–8 and Y-8–8 as disomic sperm; X-Y-8–8 as diploid sperm resulting from first meiotic division suppression; X-X-8–8 and Y-Y-8–8 as autodiploid sperm resulting from second meiotic division suppression. Strict scoring criteria were applied which accepted hyperhaploid sperm only if the two domains of the colour within a sperm were of similar size and intensity and were separated by a distance of more than half the diameter of one domain. The frequencies of sperm with hypohaploid phenotypes (X-0, Y-0 and 0–8) were scored but not included in the statistical analysis, because some chromosome loss is probably accounted for by technical artifacts (Parry et al., 1995).
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Results |
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The data from the in vitro assay for spindle disturbances using V79 cells are shown in Table I
. Trichlorfon caused a concentration-dependent increase in the percentage of abnormal mitotic figures (Figure 1). At 120 µg/ml trichlorfon, the incidence of mitoses with spindle disturbances was increased 26-fold above that found in untreated cultures and 21-fold above that found in DMSO-treated cultures. The vast majority of these spindle disturbances could be characterized as full c-mitoses showing severe disarrangement of the chromosomes, such as complete scattering in the cytoplasm, often accompanied by abnormal contractions. Initial c-mitoses characterized by improper alignment of one or more chromosomes onto the metaphase plate or the appearance of ball metaphases was less frequently observed. At the highest concentration of trichlorfon, there was a detectable effect on chromosome shape with stickiness or pycnosis indicating a cytotoxic effect. Polyploid cells were no more frequent in trichlorfon-treated cultures than in controls. Cells with anaphase and telophase bridges were not significantly more frequent in trichlorfon-treated cultures than in control cultures, indicating that chromosome exchanges were not induced. The mitotic index (MI) was clearly decreased between 40 and 100 µg/ml trichlorfon (Figure 2). At 120 µg/ml, the MI was back to control levels, which coincided with a dramatic increase in the frequency of c-mitoses.
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The patterns of epididymal mouse sperm visualized by multicolour FISH and examples of normal haploid, hyperhaploid (disomic) and diploid sperm are illustrated in Figure 3
. The numerical aberrations in target sperm chromosomes are easily viewed due to colourful fluorescent domains within the sperm. The three applied DNA probes are identified by a yellowish white domain for the X chromosome (labelled with Cy3 + FITC), a large green domain for the Y chromosome (labelled with FITC) and a red domain for chromosome 8 (labelled with CY3).
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The data from the sperm aneuploidy study with multicolour FISH are shown in Table II
. A total of 401 152 epididymal sperm were evaluated from 30 animals. The ratio of X to Y carrying sperm (X-8 versus Y-8) was 1 for all mice, as expected. No significant differences were found between the control groups for any of the abnormal sperm classes. Diploidy occurred at a very low rate in treated and control animals. The dose-dependent increase in disomic sperm is illustrated in Figure 4. After administration of trichlorfon at single doses of 200, 300 and 405 mg/kg, the frequencies of disomic sperm were 0.068, 0.074 and 0.134%, respectively. Compared with the corresponding control values of 0.046, 0.042 and 0.056%, respectively, the frequencies of disomic sperm in the three treated groups were significantly increased by factors of 1.5, 1.8 and 2.4, respectively. The frequency of X-Y-8 sperm was lower than the frequencies of X-X-8 and Y-Y-8 sperm in treated and control groups.
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The frequencies of sperm disomic for the sex chromosomes and for chromosome 8 are summarized in Table III
. The frequencies of sperm disomic for the sex chromosomes were increased significantly by factors of more than 2 compared with the corresponding control groups at each dose level, i.e. 0.036 versus 0.012%, 0.042 versus 0.014% and 0.060 versus 0.024%. In contrast, the frequency of sperm disomic for chromosome 8 was only significantly elevated above the concurrent control in the highest dose group, i.e. 0.074 versus 0.032%. At the lower dose levels, no significant differences were found for sperm disomic for chromosome 8 between the treated and control groups, i.e. 0.032 versus 0.034% and 0.032 versus 0.028%.
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To confirm the results within the laboratory, slides from the highest treatment and concurrent control groups were evaluated by another scorer (T.E.S.) utilizing the same multicolour FISH procedure. These data are presented in the last two columns of Table II
. The frequencies of disomies scored by the second scorer were generally lower but not significantly different from the scores of the first scorer (S.F.) (P > 0.05). The second scorer also found a significant difference between treated and control animals in the frequencies of disomies. |
Discussion |
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It is well documented that chemicals with aneugenic properties can alter the progression of cell division in both mitotic and meiotic cells (Adler, 1993
). In our study, V79 cells were utilized to analyse the perturbation of cell division by trichlorfon. The MI was clearly decreased between 40 and 100 µg/ml trichlorfon. The decrease in MI was in agreement with results of other researchers. de Kergommeaux et al. (1983) showed a drop in MI in Vicia faba root tip cells with elevated trichlorfon concentrations. With 4 h treatment at 10 000 p.p.m., nuclear buds, sticky metaphases/anaphases, micronuclei and c-mitoses were observed. Madrigal-Bujaidar et al. (1993) found a decrease in MI with increasing concentrations of trichlorfon in human lymphocytes in vitro accompanied by a cell cycle delay. The decreased MI was partly due to cytotoxicity and partly the result of cell cycle delay, which was probably caused by abnormal DNA synthesis during interphase (Madrigal-Bujaidar et al., 1993). In the present study, the obvious reversal of the MI level at 120 µg/ml was caused by a metaphase block, evidenced by the strong increase in the frequencies of c-mitoses. c-mitoses reflect the induction of spindle disturbances and are typical of the effect of the spindle poison colchicine (Ostergren, 1944). The appearance of full c-mitoses suggests complete inactivation of the spindle, with the chromosomes strongly contracted and with typical scattering of the chromosomes. The appearance of initial c-mitoses indicates incomplete inactivation of the spindle, which is unable to accomplish normal chromosome distribution (Ostergren, 1944). These findings are reliable indices of spindle-inhibiting effects (Andersen and Ronne, 1983). The data obtained in the present in vitro assay show that trichlorfon is a potent spindle poison in V79 cells.
There are two processes which give rise to aneuploidy, one is non-disjunction and the other is chromosome loss (Parry et al., 1995). Chromosome non-disjunction can result from malfunction of one of many processes involved in faithful segregation of chromosomes during mitosis or meiosis (Vig, 1993). The spindle apparatus is of central importance in the segregation of chromosomes (Bond, 1987). Non-disjunction resulting from trichlorfon exposure was found in cultured human lymphoblastoid cell lines (AHH-1 and MCL-5) using FISH with chromosome-specific probes (Doherty et al., 1996). Moreover, Yin et al. (1998) also provided evidence that trichlorfon may affect chromosome distribution by interfering with spindle formation. They showed that aberrant spindles and unaligned chromosomes were significantly increased in mouse oocytes treated in vitro and noted that this poses a high risk for non-disjunction and aneuploidy at anaphase. Induction of chromosome loss by trichlorfon was also shown in vitro. Doherty et al. (1996) examined trichlorfon-induced micronuclei in lymphoblastoid cell lines by FISH using a pan-centromeric probe and found a high incidence of micronuclei with centromeric signals at concentrations up to 20 µg/ml. At 80 µg/ml, micronuclei formed by acentric fragments prevailed.
The induction of non-disjunction and chromosome loss by trichlorfon discussed above suggests that trichlorfon may well induce aneuploidy in mammalian germ cells in vivo. This was confirmed by the induction of disomic mouse sperm as detected by multicolour FISH in the present study.
An important advancement in sperm cytogenetics for detecting aneuploidy was the introduction of FISH with chromosome-specific DNA probes (Wyrobek et al., 1990; Robbins et al., 1995). The use of multicolour FISH techniques made it possible to simultaneously study hyperhaploidy (disomy) for several chromosomes and to distinguish between diploid and disomic sperm (Spriggs et al., 1995, 1996; Robbins et al., 1997). Multicolour FISH methods have been developed for aneuploidy studies in mouse sperm (Wyrobek et al., 1994; Lowe et al., 1995, 1996; Adler et al., 1996), in rat sperm (Lowe et al., 1998) and in human sperm (Pieters et al., 1990; Martin et al., 1995; Spriggs et al., 1995; Robbins et al., 1993a,b; Chevret et al., 1995; Scarpato et al., 1998). For the first time, the sperm FISH assay is able to bridge the gap between experimental animals and humans (Wyrobek, 1993; Wyrobek and Adler, 1996).
In the present study, multicolour FISH using specific DNA probes for chromosomes X, Y and 8 was employed to investigate the induction of diploid and disomic sperm by trichlorfon treatment of mouse spermatocytes in vivo. In order to avoid an age effect, which has been shown to be associated with increased aneuploidy rates in sperm of mice (Lowe et al., 1995) and of humans (Griffin et al., 1995; Robbins et al., 1995), all mice used in the present investigation were young adults. The frequencies of disomic sperm were significantly higher after trichlorfon treatment than in the concurrent controls and a dose–response effect was observed. No induction of diploid sperm was noted.
Evidence suggests that different chromosomes show different susceptibilities to meiotic non-disjunction. Chromosomal differences in length, centromere position, pericentromeric and other repetitive sequences, recombination patterns and chromatin characteristics might all be related to a different susceptibility to non-disjunction (Warburton and Kinney, 1996). A different susceptibility to malsegregation between sex chromosomes and chromosome 8 was also suggested from the data in the present study. The frequency of sperm disomic for the sex chromosomes was significantly increased at the lowest dose whereas the frequency of sperm disomic for chromosome 8 showed a significant difference only in the highest dose group.
The dimorphic nature of the sex chromosomes makes it possible to distinguish disomic sperm arising at meiosis I from those arising at meiosis II. The former will have an X-Y sex chromosome constitution and the latter will be either X-X or Y-Y (Williams et al., 1993). The predominance of X-X-8 and Y-Y-8 sperm in the present study indicated that trichlorfon affected segregation at meiosis II. This observation is consistent with the report of Czeizel et al. (1993). The origin of non-disjunction was identified in two out of four cases of Down's syndrome, all with maternal exposure to trichlorfon during the critical period, and the authors demonstrated an error in maternal meiosis II in both cases. It is interesting to note in the present study that diploid sperm with the phenotype X-X-8–8 and Y-Y-8–8 were not significantly more frequent in the treated groups than in the concurrent controls. This indicates that no complete meiotic arrest was induced by trichlorfon. It also suggests that the mechanism of aneuploidy induction by trichlorfon is different from that of diazepam and similar to that of colchicine, both of which were investigated by the same sperm FISH method (Schmid et al., 1999).
The aneuploidy induction by trichlorfon observed here as well as the possible mechanism discussed above suggests that trichlorfon can be regarded as a germ cell aneugen in vivo. These results, together with the results from in vitro oocyte studies (Yin et al., 1998), provide experimental support for the conclusions by Czeizel et al. (1993) that trichlorfon exposure was causally related to the occurrence of congenital abnormality clusters in a Hungarian village.
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Acknowledgments |
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We thank Helga Gonda, Martin Skerhut and Isa Otten for their assistance with treatment of the mice and preparing and coding the slides. We also thank the BMBF, Germany, for the grant obtained by Sun Fengyun as a guest scientist from the People's Republic of China. This work was supported by EU contract no. ENV4-CT97-0471.
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Notes |
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* To whom correspondence should be addressed. Tel: +49 89 3187 2302; Fax: +49 89 3187 2210; Email:adler{at}gsf.de
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References |
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Received on February 23, 1999; accepted on August 12, 1999.
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