Mutagenesis vol. 19 no. 5 © UK Environmental Mutagen Society 2004; all rights reserved.
Tripterygium hypoglaucum (level) Hutch induces aneuploidy of chromosome 8 in mouse bone marrow cells and sperm
1State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Sciences, Fudan University, Shanghai, 200433, People's Republic of China and 2School of Life Sciences, Yunnan Normal University, Kunming, Yunnan, 650092, People's Republic of China
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Aneuploidy of mouse chromosome 8 induced by a Chinese medicinal herb, Tripterygium hypoglaucum (level) Hutch (THH) was investigated by fluorescence in situ hybridization (FISH) in vivo. Male mice were treated with THH (single i.p. injection) at doses of 120, 240 and 480 mg/kg. Colchicine (COL, 1.5 mg/kg i.p.) was used as a positive control. Bone marrow cells and epididymal sperm were collected 24 h and 22 days after treatment, respectively. Chromosome 8 aneuploidies induced by THH in bone marrow cells and sperm were determined by FISH with a biotin-16–dUTP labelled DNA probe corresponding to the centromeric region of chromosome 8. The hybridized probe was detected with avidin–FITC. The frequencies of trisomy 8 in bone marrow cells were 0.16% in the solvent control group, 0.39% in the COL-treated group and 0.33, 0.41 and 0.41% in the THH-treated groups, respectively. The frequencies of disomy 8 sperm were 0.11% in the solvent control group, 0.27% in the COL-treated group and 0.23, 0.27 and 0.27% in the THH-treated groups, respectively. The experiment showed that induced aneuploidy frequencies were higher in bone marrow cells than in sperm with COL and the two higher doses of THH (P < 0.05). All groups were significantly different from the corresponding solvent controls (P < 0.01–0.001), but there was no dose-related increase in either cell type. Considering the present results together with our previous studies, it appears that THH is a potent mammalian aneugen which may pose a genetic risk to human patients.
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Introduction |
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Aneuploidy is an important genetic change associated with birth defects, infertility, spontaneous abortions and cancer development (Li et al., 2000
; Hassold and Hunt, 2001; Parry et al., 2002). It is, therefore, imperative that any increase in its frequency due to chemical exposures should be recognized and controlled. Fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes has been increasingly utilized for the identification of aneuploidy in mammalian cells. It can be used for the rapid and sensitive detection of aneuploidy in different tissues and cell types (Pinkel et al., 1986; Eastmond et al., 1995; Robbins et al., 1995; Wyrobek and Adler, 1996; Schmid et al., 1999; Balakrishnan et al., 2002). The availability of chromosome-specific centromere probes and FISH allows the detection of aneuploidy of specific chromosomes induced by exogenous agents in mammalian sperm and various kinds of interphase cells (Wyrobek et al., 1990; Robbins et al., 1995; Schmid et al., 1999; Adler et al., 2002).
The study of compounds of plant origin has generated great interest in the fields of food and medicine (Kim et al., 1998). Many medicinal plant extracts have chemo-preventive properties and they need to be tested for their efficiency and for possible adverse effects. Tripterygium hypoglucum (level) Hutch (THH) is a traditional Chinese medicinal herb that belongs to the family Celastraceae. A water extract of THH contains alkaloids, dulcitols, terpenes, lactones and pigments. The dry root can be immediately used in the form of a decoction with water or, alternatively, the unpurified water extract can be compacted to make tablets for clinical use. THH has been used widely for the treatment of various human immunological diseases, such as systemic lupus erythematosus or rheumatoid arthritis. It has been reported that the use of THH leads to reversible inhibition of germinal cell development in both humans and rats (Luo et al., 1988; Zhou et al., 1991). In our previous studies THH showed aneugenic activity in mouse bone marrow cells in vivo, as shown by the C-mitotic effect assay, by chromosome structural aberration analysis and by the micronucleus assay (Wang et al., 1993). Also, THH induced germinal aneuploidy in Drosophila melanogaster (Wang and He, 1995). Wang and Yang reported that THH significantly increased kinetochore-positive micronuclei in mouse bone marrow and 3T3 cells (Wang et al., 1994a; Yang and Cao, 2001). However, no information exists regarding THH-induced specific chromosome aneuploidy in either somatic or germinal cells.
In the present study the aneugenic activity of THH in mice was analyzed in bone marrow cells and sperm by the FISH assay with a DNA probe for chromosome 8.
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Animals
Experiments were performed with 10–12-week-old Kunming male mice, weighing 22–28 g. Each experimental dose group consisted of eight animals. Eight solvent control males were used in each experiment. Another eight animals were treated with colchicine (COL) as a positive control.
Chemical and THH preparation
COL was purchased from Serva (Japan) and dissolved in sterile distilled water before use.
The dry root of THH was provided by Kunming Medicine Co. (Yunnan, China). A sample of 50 g of mashed THH was kept in 500 ml of distilled water for 2 h and then boiled for 10 min and allowed to cool to room temperature for 30 min. The procedure was repeated three times to ensure maximum extraction. The supernatant was filtered and lyophilized. A sample of 2.4 g lyophilized THH extract was obtained and the test doses were prepared from this extract. THH extract was redissolved in sterile distilled water before use according to the doses required. The water extract of THH contains alkaloids, dulcitols, terpenes, lactones and pigments.
Treatment
The mice were treated with 120, 240 or 480 mg/kg THH or 1.5 mg/kg COL once by i.p. injection. The injected volume was 0.1 ml/10 g body wt. The maximum dose of THH was 50% of the LD50, which was the lowest dose shown to induce mitotic arrest in mice (Wang et al., 1994b). The lowest dose was within the range of human clinical use (water extract of 3–9 g dry root/day) when the decoction was used immediately (Jiangsu New Medicine College, 1986).
Preparation of bone marrow leukocytes and DNA hybridization
All animals in the test groups and the two controls were killed 24 h after treatment. The sampling interval was chosen on the basis of a previous study (Wang et al., 1993), which showed that 24 h is the most sensitive time for aneuploidy induction by THH. Slide preparation followed the methods described by Adler (Adler, 1984). Briefly, the animals were killed by cervical dislocation and bone marrow was flushed from both femora into centrifuge tubes with fetal bovine serum. Hypotonic treatment was carried out with 0.75% KCl for 10 min at room temperature. Cells were centrifuged at 1000 r.p.m. for 5 min and fixed twice with cold methanol for 10 min. Cell suspensions were dropped gently onto microscope slides. The slides were air dried and stored at –20°C until processed for hybridization.
The chromosome-specific probe for chromosome 8 (Boyle and Ward, 1992) was a generous gift from Dr Adler (GSF–Institute of Experimental Genetics, Germany). Plasmid DNA for chromosome 8 (clones 8-4a and 8-5e) were transformed in Escherichia coli XL1-blue and were isolated using a Wizard Plus Midipreps DNA Purification System (Promega Corp.). The probe was labeled with biotin-16–dUTP (Boehringer, Mannheim, Germany) using a nick translation system (Gibco BRL, USA).
Prior to in situ hybridization the slides were pretreated with pepsin (0.005% in 0.01 N HCl) at 37°C for 5 min. Slides were then washed twice in phosphate-buffered saline (PBS) (pH 7.0, 5 min each) at room temperature. Hybridizations were performed according to a modified technique of Pinkel et al. (1986, 1988). Slides were denatured at 76°C for 8 min in prewarmed 70% formamide in 2x SSC and then dehydrated through a cold ethanol series (70, 90 and 100%, 2 min each) at 4°C. Labelled DNA probe was mixed with Master Mix 2.1 (55% formamide, 10% dextran in 1x SSC) and denatured at 76°C for 10 min, then added to the slides, which were coverslipped and sealed with rubber cement. Hybridization was carried out overnight in a moist chamber at 37°C. Post-hybridization washing consisted of two steps: 30 min in 50% formamide in 2x SSC (pH 7.0) at 45°C and 30 min in PN buffer (50 mM sodium phosphate buffer, pH 8.0, containing 300 mM NaCl) at 37°C. The hybridized probe was detected with avidin–FITC (1:400) (Sigma) for 30 min in the dark followed by washing in PN buffer at 45°C for 5 min. The nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (0.1 µg/ml in PBS) for 10 min at room temperature. Slides were coverslipped in Vectashield mounting medium (Vector Laboratories), sealed with nail varnish and kept at –4°C in the dark until microscopic analysis.
Preparation of epididymal sperm and hybridization
All animals in the test groups and the two solvent control groups were killed 22 days after application of the test chemicals or solvent. Sperm were collected from the caudae epididymes according to the description by Schmid et al. (1999). The interval of 22 days allowed spermatocytes exposed during meiosis to develop into mature sperm (Oakberg, 1956; Adler, 1996). The techniques for preparation and decondensation of epididymal sperm and preparation of the DNA probes and the hybridization conditions were described earlier (Adler et al., 1996; Lowe et al., 1996; Schmid et al., 1999). Briefly, both epididymes were dissected, placed in a Petri dish, prepared and incisions were made. Subsequently, they were individually placed in an Eppendorf cup filled with 300 µl of fetal calf serum. The cups were placed on an Eppendorf incubator at 32°C for 30 min to allow the sperm to actively leave the epididymes. The tissues were removed from the cups and the sperm suspensions were stored at 4°C overnight. Aliquots of 5 µl of sperm suspension were pipetted onto clean, dry glass slides. The unfixed cells were spread across the slide and allowed to dry overnight. The slides were stored at –20°C.
Prior to hybridization the slides were incubated in a Coplin jar in 10 mM dithiothreitol (Sigma) and then in 4 mM lithium-3,5-diiodosalicylic acid (Sigma) for 60 min at room temperature. The slides were air dried for hybridization.
The procedures for labelling the probe were the same as mentioned above.
Hybridizations were performed according to Pinkel et al. (1986, 1988) with minor modifications. Labelled DNA probe was mixed with Master Mix 2.1 and denatured at 78°C for 8 min. The sperm slides were denatured in 70% formamide (in 2x SSC, pH 7.0) at 78°C for 5 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 mixture.
Hybridization was carried out overnight 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. Probe detection was the same as in bone marrow cells described above.
Scoring
A total of 2000 leukocytes or 4000 sperm per individual were scored depending on the quality of hybridization. A Nikon E400 fluorescence microscope was used with a triple band filter (DAPI, FITC and Rhodamine; Nikon), allowing simultaneous visualization of the fluorochromes used. Only correctly hybridized cells were considered. A normal diploid bone marrow cell was expected to contain two green signals in its nucleus. A nucleus with one green signal was considered monosomic for chromosome 8 and one with three hybridization signals as trisomic. A normal sperm was expected to contain one single green signal, since sperm cells are haploid. Sperm with two green signals were regarded as disomic for chromosome 8. Since only one DNA probe was used, disomic and diploid sperm could not be differentiated. Only hyperploidy was considered as aneuploidy in our study. Monosomic leukocytes and nullsomic sperm were counted but not included in the statistical evaluation because they may be caused by technical artifacts.
Cells were scored as having two domains when signals were of similar sizes and intensities and were separated by a distance of more than half the diameter of one domain.
Statistical analyses
The 
2 test with Yates' correction was used to determine significant differences between control and individual experimental groups.
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Results |
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FISH in mouse bone marrow leukocytes
The individual animal counts of bone marrow cells with three signals for chromosome 8 are shown in Table I. A summary of the results with the solvent control, the positive control chemical (COL) and the test compound THH is given in Table II. COL significantly increased the frequency of trisomic cells as expected. Significant increases in trisomic cells were observed in all THH-treated groups (P < 0.01). In all test groups the frequencies of trisomic cells were more than twice the frequency in the solvent control (0.33–0.41 versus 0.16%, respectively). No dose-related increase was found with THH. The relative aneugenic potencies of 1.5 mg/kg COL and all doses of THH were similar.
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FISH in mouse sperm
Table III shows the individual animal counts of sperm with two signals for chromosome 8. Table IV gives the average frequencies of sperm with chromosome 8 disomy at 22 days after treatment with COL or THH. The frequencies of disomic sperm were increased significantly in all treatment groups by more than twice the control (0.23–0.27 versus 0.11%, P < 0.001). However, no dose–response effect was found. The relative aneugenic potencies of 1.5 mg/kg COL and the two higher doses of THH were identical.
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The average frequency of sperm with no green fluorescent signal was 0.24% (0.13–0.29%). Since this frequency is of the same order as the disomy frequency, it is argued that the sperm without signals represent real nullisomic sperm and that the hybridization was of high quality.
The values for bone marrow leukocytes with three fluorescent signals (trisomies) and for sperm with two fluorescent signals (disomies and diploidies) are compared in Figure 1. The percentages of aneuploid bone marrow leukocytes were higher than those of aneuploid sperm for the two upper THH dose groups and for the positive control group (P < 0.05).
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Discussion |
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Using the FISH method with a single chromosome-specific DNA probe, the present study was performed to investigate THH-induced aneuploidy in mouse bone marrow cells and sperm. The results from our study support the hypothesis that THH is a potent aneugen in both mammalian somatic cells and germ cells.
In this study the frequencies of aneuploidies (trisomic leukocytes and disomic sperm) may be considered to be approximations of aneuploidy frequencies, since FISH with a single DNA probe is unable to distinguish hyperploidy from polyploidy. In bone marrow the frequencies of cells with three signals may theoretically include some triploidies induced by THH. However, this is considered a very rare event caused by unequal cell division. Cells with four signals, which would represent tetraploidies caused by suppression of cell division, were not observed. They would have been expected since our previous cell cycle examination of bromodeoxyuridine-labeled bone marrow cells demonstrated that 480 mg/kg THH induced mitotic inhibition (Wang et al., 1993, 1994b). The frequencies of sperm with two signals, on the other hand, may include some diploidies induced by THH. This is more likely because THH has been reported to reversibly inhibit spermatogenesis (Zhou et al., 1991), which may be accompanied by suppression of meiotic cell division.
In the present experiment the aneuploidy baseline for chromosome 8 in mouse sperm was higher than that reported by other authors (Schmid et al., 1999; Shi et al., 1999; Adler et al., 2002). We suggest that this indicates that our albino Kunming strain of mice exhibits higher genomic instability.
The present results show that the aneuploidy frequencies induced by THH were higher in mouse bone marrow than in sperm. This is in contrast to the observation for carbendazim by de Stoppelaar et al. (1999) in rats. In that study, the peripheral blood micronucleus assay was a less sensitive indicator for the aneugenic potential of carbendazim than the epididymal sperm aneuploidy/diploidy assay. This may be due to the different specific actions of the test compounds. However, we conclude that the assessment of aneuploidy induction in two different cell types by the same technology gives a more reliable estimate.
THH is used therapeutically both in the form of tablets and as a decoction of the original dry root. The latter form is usually employed in traditional Chinese medicine and is prepared in the same way as in our experiments. The adult dose is normally an extract of 3–9 g dry root/day (Jiangsu Medical College, 1986). The lowest dose in the present experiments is in the range of the clinical dose for humans. The present results corroborate the aneugenic potential already indicated by published work (Wang et al., 1993, 1994a,b; Wang and He, 1995) and refine the genotoxic characteristics of THH. Therefore, we conclude that THH is not only a potent aneugen in mouse somatic and germ cells, but may also have the same activity in humans. It seems important for Chinese medical professionals to consider a possible genetic risk of THH and to cautiously advise patients to abstain from reproduction for 6 months after the end of treatment with THH.
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Acknowledgments |
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This study was supported by the National Natural Science Foundation of China (39860035 and 30160038) and the International Scientific Cooperation Project of Yunnan. We are very thankful to Drs I.-D.Adler and T.E.Schmid for their generous supply of the chromosome probe. We are also grateful to Dr Adler for helping to revise the manuscript.
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3 To whom correspondence should be addressed. Tel: +86 21 65649899; Fax: +86 21 65649899; Email: jlxue{at}fudan.ac.cn
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Received on March 18, 2004; revised on July 8, 2004; accepted on July 12, 2004.
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