Mutagenesis, Vol. 14, No. 1, 51-56,
January 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
Noscapine hydrochloride disrupts the mitotic spindle in mammalian cells and induces aneuploidy as well as polyploidy in cultured human lymphocytes
Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521 and 1 Pfizer Inc., Groton, CT 06340, USA
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Noscapine hydrochloride is a centrally acting antitussive opium derivative widely used in cough suppressants. Recent studies have reported that noscapine is a potent inducer of polyploidy but not of aneuploidy in vitro. To obtain more comprehensive information about the cytogenetic effects of this compound, we treated cultured human lymphocytes (HPL) and Chinese hamster ovary (CHO) cells with various concentrations of noscapine hydrochloride. Using a differential staining technique noscapine was shown to disrupt the mitotic spindle at concentrations <5 µg/ml in both cell types. The use of multicolor fluorescence in situ hybridization (FISH) on noscapine-treated human lymphocytes showed a dose-dependent induction of hyperdiploidy of chromosome 1 but not of chromosomal breakage in the 1cen–q12 region under in vitro conditions, indicating that noscapine is specifically inducing numerical chromosomal aberrations. FISH with probes targeting different chromosomes revealed that noscapine is capable of inducing both polyploidy and true hyperdiploidy. Our results show that noscapine, by disrupting the function of the mitotic spindle, has the ability to induce aneuploidy and not uniquely polyploidy as previously reported. By using these types of molecular cytogenetic techniques, it should be possible to evaluate the ability of noscapine to induce aneuploidy in the upper intestinal tract in vivo.
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Introduction |
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Noscapine is an isoquinoline alkaloid obtained from opium, where it occurs in concentrations ranging from 4 to 10%. It was first isolated by Roboquet in 1817, who named it narcotine (Nayak et al., 1965
). It is a non-narcotic, centrally acting, antitussive drug and is used in a wide variety of cough suppressants (Gatehouse et al., 1991).
Recent in vitro studies have raised questions about the safety of this compound. Noscapine has been shown to induce high levels of polyploidy in cultured Chinese hamster lung cells (Ishidate, 1988). These results were confirmed by Gatehouse et al. (1991), who found large increases in polyploidy in V79 and cultured human lymphocytes (HPL) treated for 24 or 48 h over a dose range of 15–120 µg/ml. In addition, these authors observed spindle damage in V79 cells and human skin fibroblasts and found chromosomes dislocated from the mitotic spindle, indicating a direct effect upon the spindle and/or its function. In the same year Mitchell et al. (1991) found that noscapine induces high levels of polyploidy in HPL after a 24 h treatment but not after a 4 h treatment. Noscapine has also been reported to induce morphological transformation of immortal Syrian hamster dermal fibroblasts in vitro over a concentration range of 30–120 µg/ml and it was shown that the transformed colonies had an increase in chromosome number and a non-random duplication of a translocated chromosome 9 (Porter et al., 1992). However, the available in vivo data have not confirmed these in vitro results. Noscapine did not increase the frequency of micronucleated polychromatic erythrocytes in mouse bone marrow (Furukawa et al., 1989) and in mouse oocytes no increase in the frequency of hyperdiploid or polyploid cells was detected (Tiveron et al., 1993). The negative results seen in vivo are probably due to rapid metabolism and elimination of noscapine and its low bioavailability (Nayak et al., 1965; Gibaldi and Weiner, 1966; Dahlstrom et al., 1982; Karlson et al., 1990).
A variety of recently developed tools are available for the detection of structural and numerical chromosomal alterations. Fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes is increasingly utilized for the detection of chromosome aberrations induced in vitro and in vivo by physical and chemical agents. We have shown that FISH with chromosome-specific DNA probes can be used to detect hyperdiploidy and polyploidy in vitro and in vivo (Eastmond and Pinkel, 1990; Eastmond et al., 1994; Hasegawa et al., 1995; Rupa et al., 1995, 1997). One of the major problems of interphase FISH with a single chromosome-specific DNA probe is that the analysis cannot distinguish between hyperdiploidy and polyploidy. For risk assessment this could be critical because it is not clear whether or not polyploidy is an important genetic event (Mitchell et al., 1995). We have recently shown that multicolor FISH with 
-satellite probes for chromosomes 1, 7 and 9 can be used to distinguish between hyperdiploidy and polyploidy in vitro (Schuler et al., 1998).
In this paper we describe the different cytogenetic effects of noscapine in HPL and the effects of noscapine on the mitotic spindle in both HPL and Chinese hamster ovary (CHO) cells. Multicolor FISH with two adjacent or tandem labeling probes for chromosome 1 was used to estimate the effect of noscapine on the induction of hyperdiploidy and chromosomal breakage in the 1cen–q12 region of HPL. Another multicolor FISH technique with -satellite probes for chromosome 1, 7 and 9 was used to estimate the proportion of cells exhibiting real hyperdiploidy and polyploidy.
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Material and methods |
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Chemicals
Noscapine hydrochloride (CAS no. 128-62-1) was purchased from Sigma Chemical Co. (St Louis, MO).
Cell culture
CHO cells (CHO-K1-BH4) were obtained from the Vermont Regional Cancer Center (Burlington, VT) and were cultured in Ham's F12 medium (Gibco BRL, Grand Island, NY) with 5% fetal bovine serum (Gibco BRL), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (Gibco BRL). Peripheral blood was collected from healthy volunteers in heparinized vacutainers. Slightly different culturing procedures were used reflecting the standard conditions of the two laboratories participating in this study. Whole blood cultures of human lymphocytes for the spindle assay were cultured in William's medium E (Gibco BRL) supplemented with 5% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 1% (v/v) phytohemagglutinin type M solution (Gibco BRL). Isolated lymphocytes cultures were established in RPMI 1640 (Gibco BRL) medium supplemented with 5% fetal calf serum (Hyclone, Logan, UT), 5% iron-supplemented calf serum (Hyclone), 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin (Mediatech, Washington, DC) and 2.36% phytohemagglutinin type M solution (Gibco BRL). All cells were maintained at 37°C in 5% CO2 and 100% humidity.
Spindle assay
Spindle assays were performed using the method developed by Wissinger et al. (1981) with minor modifications. CHO cells were grown as exponential growing monolayers in chambers (Nunc Flask) directly on slides. Immediately prior to treatment, the medium was removed from cultures and replaced by medium containing noscapine in 1% dimethyl sulfoxide (DMSO). After 24 h, the CHO cells were harvested and fixed with 3:1 (v/v) methanol/acetic acid supplemented with 2 mM calcium chloride and 4 mM magnesium chloride for 30 min at room temperature. The fixation step was repeated with fresh fixative for another 60 min and the slides were air dried.
HPL at a cell density of 3.5–4.5x105 cells/ml were treated with noscapine 48 h after culture initiation. The final DMSO concentration was 1%. At the end of the 24 h treatment period, cells were centrifuged and fixed as described above. Cell suspensions were dropped onto precleaned glass slides and air dried.
The cells were then treated with a 5% perchloric acid solution, rinsed in distilled water and air dried. The cells were stained with a solution of 0.5% safranin O (Sigma) and brilliant blue R (Sigma) in 10% acetic acid. Slides were rinsed with distilled water, air dried and mounted with Accu-mount 60 (Stephens Scientific, Riverdale, NJ). All scoring and classification was done at high magnification (630 or 1000x) using a Zeiss Axiophot with brightfield illumination and phase contrast. Only intact cells with chromosomes in the appropriate orientation were included in the evaluation.
At least 200 mitotic cells from two separate cultures were classified by mitotic stage (metaphase, anaphase or telophase) and further analyzed for spindle abnormalities and chromosome lagging or bridging in anaphase or telophase. Metaphase cells were evaluated for spindle integrity and abnormal cells were classified as follows: (i) normal bipolar cells having two poles, with all chromosomes aligned properly at the metaphase plate; (ii) abnormal bipolar cells containing chromosomes which were not attached to spindles and/or not properly aligned on the metaphase plate; (iii) abnormal polar configurations containing apolar (no poles), monopolar (one pole) or multipolar (>2 poles) cells.
Multicolor fluorescence in situ hybridization (FISH)
To determine the effect of noscapine on the induction of hyperdiploidy, polyploidy and chromosomal breakage in the 1cen–q12 region, we treated isolated human lymphocytes with noscapine 24 h after culture initiation and harvested the cells 48 h later using standard procedures (Eastmond et al., 1994). Briefly, cells were treated with 75 mM KCl for 30 min at room temperature then fixed several times with 3:1 (v/v) methanol/acetic acid, dropped onto precleaned glass slides and air dried. Slides were stored at –20°C in nitrogen-containing storage bags until use.
Detailed protocols of probes, labeling and hybridization conditions for the tandem labeling procedure for chromosome 1 and the principle underlying this technique are described in detail elsewhere (Rupa et al., 1995). To simultaneously label the centromeric regions of chromosomes 1, 7 and 9, specific -satellite probes for chromosomes 7 and 9 were generated by PCR with genomic DNA and oligonucleotide primers. Probe generation for the -satellite region of chromosome 9 is described in Hasegawa et al. (1995). For the generation of a chromosome 7-specific -satellite probe we used a specific chromosome 7 -satellite 20mer primer (Pellestor et al., 1995) designated Asat7c (5'-AGC GAT TTG AGG ACA ATT CT-3') and a human all centromere-specific 30mer primer (Meyne et al., 1989) named SO-AllCen (5'-GTT TTG AAA CAC TCT TTT TGT AGA ATC TGC-3'). As a template, we used DNA from the somatic cell hybrid GM10791, a hamster cell line containing human chromosome 7 [Human Genetic Mutant Cell Repository (NIGMS), Coriell Institute for Medical Research, Camden, NJ]. PCR conditions were similar to those described in Hasegawa et al. (1995). After hot starting the reaction by denaturing the DNA at 94°C for 5 min and then adding 5 U Tfl polymerase (Epicenter Technologies, Madison, WI), amplification was performed for 30 cycles of 30 s at 94°C, 30 s at 42°C and 1 min at 72°C, followed by one final cycle of 15 min at 72°C. PCR amplification products were nick-translated according to the protocol provided with the DNA polymerase/DNase I enzyme mixture (Gibco BRL) using FluoroGreenTM (BDS, Biological Detection Systems Inc., Pittsburg, PA) as the label for chromosome 7 and digoxigenin (Boehringer Mannheim, Indianapolis, IN) for chromosome 9.
For all in situ hybridizations, standard conditions were used (Trask and Pinkel, 1990). Triple labeling hybridizations with -satellite probes for chromosomes 1, 7 and 9 were performed in 55% formamide, 10% dextran sulfate, 1x SSC using 1 µg sheared herring sperm DNA, 0.5 µl biotin-labeled -satellite probe for chromosome 1 (Oncor Inc., Gaithersburg, MD), 20–100 ng digoxigenin-labeled -satellite for chromosome 9, 20–100 ng FluoroGreenTM-labeled -satellite probe for chromosome 7 and deionized water as required, all in a volume of 10 µl. Post-hybridization washes were performed once in 2x SSC, three times in 60% formamide, 2xSSC and once in 2x SSC for 5 min, all at 42–44°C. Biotinylated and digoxigenin-labeled probes were simultaneously labeled using Cascade BlueTM avidin [Molecular Probes Inc., Eugene, OR; 20 µg/ml in PN buffer (0.1 M phosphate buffer, pH 8.0, 0.5% w/v Triton X-100) with 5% non-fat dry milk (PXM)] and a mouse anti-digoxigenin IgG (Boehringer Mannheim; 1.6 µg/ml PXM), followed by an amplification round with biotinylated anti-avidin IgG (Vector Laboratories, Burlingame, CA; 2.5 µg/ml) and digoxigenin-conjugated sheep anti-mouse antibody (Boehringer Mannheim; 10 µg/ml PXM). A third layer of the affinity reagents, Cascade BlueTM (20 µg/ml in PXM) and rhodamine-conjugated sheep anti-digoxigenin IgG (Boehringer Mannheim; 10 µg/ml in PXM) was applied. Nuclei were counterstained for 10 min with Yoyo-1 (Molecular Probes Inc., Eugene, OR; 30 nM in PBS) and mounted with a phenylenediamine antifade medium.
All slides were scored using a Nikon fluorescence microscope at 1250x magnification. Scoring was performed on coded slides with a minimum of 1000 cells/dose evaluated for each of two separate experiments. Fluorescence filters and scoring criteria for the tandem labeling technique were described earlier (Eastmond et al., 1994). For triple labeling slides counterstained with Yoyo-1, a triple bandpass filter (Chroma Technology Corp., Brattleboro, VT; no. P/N 61002) was used to simultaneously visualize the yellow-green (FluoroGreenTM; -satellite chromosome 7), red (rhodamine; -satellite chromosome 9), blue (Cascade BlueTM; -satellite chromosome 1) and dark green (Yoyo-1 counterstain). Nuclei were examined initially for the number of red and yellow-green signals only. In the case of a cell with more than two red and/or yellow-green signals, the presence of more than two signals for chromosome 1 and the signals for chromosomes 7 and 9 were verified by changing to a filter optimal for the individual fluorochrome: a blue filter (Nikon B-2A, excitation at 450–490 nm, emission at 520 nm) for the FluoroGreenTM signals, a green filter (Chroma Technology Corp., Brattleboro, VT; no. 31004, excitation at 540–580 nm, emission at 600–660 nm) for the rhodamine signals and a UV filter (Nikon UV-1A, excitation at 330–380 nm, emission at 420 nm) for the Cascade BlueTM signals. Cells with more than two signals for only one chromosome were classified as hyperdiploid for this chromosome, cells with more than two signals for two chromosomes were classified as `possible polyploid' and cells with three or more signals for all three chromosomes were classified as polyploid.
Statistical analysis
Dose-related increases in the frequency of spindle damage, hyperdiploidy, chromosomal breakage and polyploidy induced by noscapine were determined using the Cochran Armitage test for trend in binomial proportions (Margolin et al., 1986). Following a positive response in the trend test, a one-tailed Fisher exact test was used to compare each treatment with the respective control. Critical values were determined using a 0.05 probability of type I error.
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Spindle damage
Illustrations of the effects of noscapine on the mitotic spindle in CHO cells and HPL are shown in Figure 1
. Photographs of the various normal mitotic stages in the CHO cells are presented in Figure 1a–c. Examples of spindle damage induced by noscapine in this cell line are seen in Figure 1d–i. An apolar metaphase and a monopolar metaphase are shown in Figure 1d and e, respectively. In both cases, no equatorial plate can be detected and the chromosomes do not appear to be attached to a mitotic spindle. In comparison, the bipolar metaphase in Figure 1f displays a well-developed equatorial plate but three chromosomes are not attached to the mitotic spindle. A typical example of a multipolar metaphase with two separate equatorial plates and misaligned chromosomes is seen in Figure 1g. Figure 1h presents an anaphase cell with a lagging chromosome and Figure 1i a telophase with a chromosome bridge. Figure 1j–l exemplifies mitotic human lymphocytes after treatment with noscapine. An example of a normal metaphase is seen in Figure 1j. The multipolar anaphase in Figure 1k contains four different equatorial plates and the anaphase in Figure 1l shows an anaphase bridge.
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Mitotic profiles obtained after treatment of CHO and HPL with noscapine are given in Figures 2a–c
and 3a–c, respectively. A significant increase in metaphases (P < 0.05, binomial trend test followed by one-tailed Fisher's exact test) among the mitotic figures combined with a decrease in anaphases and telophases, indicating a noscapine-induced mitotic arrest, was seen in CHO cells at 3.8–30 µg/ml and in HPL at 5–20 µg/ml. A treatment-related increase in the frequency of abnormal mitotic cells was seen in CHO cells (Figure 2b and c) and HPL (Figure 3b and c) at all tested concentrations. At lower concentrations, the abnormalities consisted primarily of abnormal bipolar metaphase cells containing chromosomes that were not attached to spindles or were not aligned on the metaphase plate (Figures 1f, 2c and 3c
). Dislocated or misaligned chromosomes could fail to segregate at the completion of mitosis, resulting in formation of aneuploid cells. At high concentrations, more severe damage to the mitotic spindle was seen, especially formation of apolar and monopolar spindles (Figures 2b and 3b). These types of spindle damage would be more likely related to induction of polyploidy rather than actual aneuploidy. Also seen at the high concentrations are `lagging chromosomes' in anaphase and telophase cells (Figures 2c and 3c). Chromosomes that do not migrate properly to the appropriate pole have a high likelihood of malsegregation, hence resulting in aneuploidy.
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Tandem labeling technique
The principle underlying the tandem labeling technique has been published in detail elsewhere (Eastmond et al., 1994
; Rupa et al., 1995). Using a Cy3-labeled classical satellite probe, which specifically targets the pericentric heterochromatin region located on chromosome 1, and a fluorescein-labeled -satellite probe which binds to a smaller adjacent centromeric region, this technique allows hyperdiploidy/polyploidy to be distinguished from breakage affecting the pericentric heterochromatin of chromosome 1. The frequencies of numerical and structural aberrations affecting this chromosome 1 region in HPL treated for 48 h with noscapine are presented in Figure 4. Noscapine induced a significant dose-related increase in hyperdiploidy/polyploidy but not in breakage affecting the pericentric heterochromatin region of chromosome 1. Significant increases in hyperdiploidy could be detected at noscapine concentrations between 22.5 and 157.5 µg/ml, with the highest tested dose being the most effective.
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Triple labeling technique
Because it is not possible to discriminate between hyperdiploidy and polyploidy using FISH with a probe for only one chromosome, a multicolor FISH technique with three chromosome-specific
-satellite probes was used. For convenience in scoring, only the red chromosome 9 and green chromosome 7 signals were counted using the triple bandpass filter. In the case of a cell with more than two hybridization regions for chromosome 7 and/or 9, the filter was changed to the Cascade Blue-, FITC- or rhodamine-specific filter and the number of copies for each of the three individual chromosomes was determined. The results of this approach in lymphocytes treated with 22.5 and 157.5 µg/ml noscapine are shown in Figure 5. Cells with more than two copies of one chromosome were classified as hyperdiploid cells, cells with more than two copies of two chromosomes were summarized as `possible' polyploid and cells with more than two signals for all three chromosomes were classified as polyploid. Using this approach, most abnormal nuclei were classified as polyploid and possible polyploid after treatment with noscapine. Significant increases in all three classification categories were seen at both tested noscapine concentrations, indicating that noscapine is capable of inducing hyperdiploidy as well as polyploidy.
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Discussion |
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In this study we combined spindle staining with modern molecular cytogenetic techniques to provide a more comprehensive overview of the aneuploidy and polyploidy inducing effects of the antitussive drug noscapine. The results from these in vitro studies demonstrate that noscapine induces both aneuploidy and polyploidy in HPL and that these effects are probably the result of the effects of noscapine on the mitotic spindle. Noscapine induced mitotic disturbances, hyperdiploidy and polyploidy in HPL at concentrations which can be locally achieved with therapeutic doses of noscapine.
We have recently developed a multicolor FISH approach to detect nuclei with multiple hybridization regions formed from chromosomal breakage. With this multicolor FISH application, we labeled the pericentric heterochromatin region, which is large and prone to breakage by physical and chemical agents (Brogger, 1977; Rupa et al., 1995), with the red fluorochrome Cy3 and the adjacent centromeric region, which is somewhat smaller and much less prone to breakage, was marked with a fluorescein-labeled -satellite probe. Using this approach on cultured human lymphocytes treated with noscapine, we found a significant induction of numerical aberrations but not of chromosomal breakage in the pericentric heterochromatin of chromosome 1. This is in agreement with earlier studies where noscapine has been shown to induce polyploidy in CHO cells and human lymphocytes in the absence of point mutations and chromosome breakage.
Using a multicolor FISH strategy we could show that noscapine is capable of inducing aneuploidy in cultured human lymphocytes whereas other studies only investigated the induction of polyploidy by this compound. This is important in that the biological significance of polyploidy as a genotoxic end point is still not defined, due in part to its widespread occurrence in somatic tissues such as the human myocardium, bladder and rodent liver (Keighren and West, 1993; Brodsky et al., 1994). In comparison, aneuploidy is the most significant cause of pregnancy loss as well as genetic defects among live births (Hook, 1986). Genomic imbalance produced by aneuploidy in somatic cells may also play a significant role in the development of certain tumors (Oshimura and Barrett, 1986; Hecht and Hecht, 1987; Oshimura et al., 1988). Numerous human malignancies exhibit specific chromosomal numerical changes (Heim and Mittelman, 1986) and studies of childhood malignancies such as retinoblastoma have established that loss of a tumor suppressor gene can occur through a mechanism involving chromosome imbalance (Cavenee et al., 1983).
Mitchell et al. (1991) reported that noscapine-induced hypodiploidy could be a technical artifact arising from membrane effects which then resulted in chromosomal loss during metaphase slide preparation. Although noscapine is known to react with membrane receptors (Karlson et al., 1988), artifacts from membrane effects are not likely to be responsible for the observed induction of hyperdiploidy in our study because chromosome-specific FISH on interphase cells would not detect increases in hyperdiploidy or polyploidy if this were the sole effect. In terms of effective concentrations, we observed significant increases in spindle damage at concentrations as low as 3.8 µg/ml in CHO and 2.5 µg/ml in lymphocytes, with induction of hyperdiploidy at concentrations of 15 µg/ml and higher. This indicates that disruptions in the mitotic spindle can be detected at concentrations which do not result in numerical aberrations detectable by FISH. These effective concentrations are below those expected in the upper gastrointestinal tract (typically 150 µg/ml in a 10 ml dose) but above the expected systemic concentration following absorption (Nayak et al., 1965; Dahlstrom et al., 1982). From these results, it is concluded that noscapine is unlikely to cause chromosomal changes in organs such as liver, bone marrow and ovary. Consequently, it is not surprising that noscapine was negative in the bone marrow micronucleus assay in mice (Furukawa et al., 1989) and the mouse oocyte test (Tiveron et al., 1993) in vivo. However, the ability of noscapine to induce aneuploidy in the upper intestinal tract has not been addressed and should be evaluated using an appropriate in vivo test.
In conclusion, our results show that noscapine is a potent inducer of spindle damage, hyperdiploidy and polyploidy in human lymphocytes cultures at concentrations which can be found after oral application of this drug in cough medicine. Further studies are required to clarify the aneugenic capacity of this drug in vivo and to evaluate its potential as a hazard to human health.
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2 To whom correspondence should be addressed. Tel: +1 909 787 4497; Fax: +1 909 787 3087; Email: eastmond{at}ucrac1.ucr.edu
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Received on March 18, 1998; accepted on August 13, 1998.
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