Spindle poisons can induce polyploidy by mitotic slippage and micronucleate mononucleates in the cytokinesis-block assay

Mutagenesb vol.13 no.2 pp.193-198, 1998 Spindle poisons can induce polyploidy by mitotic slippage and micronucleate mononucleates in the cytokinesis-block assay Azeddine Elhajouji1, Monica Cunha and Micheline Kirsch-Volders Laboratory for Human Genetics, Vrije Universiteit Brussel, Pleinlaan 2, 1050-Brussels, Belgium Introduction The in vitro cytokinesis-blocked micronucleus (MN) method has been shown to have many advantages. Essentially it discriminates between cells which did not divide from those which divided once or more and allows concurrent scoring of different end-points, e.g. MN, chromosome loss, chromosome non-disjunction and apoptosis (for reviews see Fenech, 1997; Kirsch-Volders et al, 1997). Therefore, this methodology has gained the favour of both research and genotoxicology laboratories: efforts are being made to introduce it into the test battery as a replacement for the in vitro chromosome aberration test and for assessment of aneuploidy induction (Aardema et al, 1998; Albertini and Kirsch-Volders, 1997; Marzin, 1997). In the many studies performed with this method almost no attention has been paid to the biological information contained in mononucleate lymphocytes. It is commonly accepted that mononucleates represent a population of cells which did not divide in culture. This assumption is a simplified one, since mononucleate cells may also derive from cells which are insensitive to cytochalasin B (Surrales et al., 1994; Zijno et al., 1994) and/or cells which were blocked in metaphase after Materials and methods Chemicals Nocodazole (NOC; CAS 31430-18-9) was provided by Janssen Pharmaceutica (Beerse, Belgium). Carbendazim (MBC; CAS 10605-21-7) was purchased from Aldrich Chemie (Steinheim, Germany) and mebendazole (MEB; CAS 31431-39-7) from Sigma Chemical Co. (Brussels, Belgium). These three chemicals were dissolved in dimethylsulfoxide (DMSO; Merck, Darmstadt, Germany) for spectroscopy. Methyl methanesulfonate (MMS; CAS 66-27-3) was purchased from Merck and dissolved in phosphate-buffered saline (PBS). Colchicine (COL; CAS 64-86-8) and mitomycin C (MMC; CAS 50-07-7) were purchased from Janssen Chemica (Beerse, Belgium) and dissolved in PBS. Cytochalasin B (Sigma Chemical Co.) was dissolved in DMSO and kept as a stock solution of 4 mg/ml at -20°C. Cultures Human peripheral blood samples were obtained from healthy volunteers not exposed to known mutagens. Blood was drawn by venipuncture and heparinized with Calparine0 (Sanofi, Labaz, France). Lymphocytes were cultured in Ham's F-10 medium supplemented with HEPES buffer (Gibco BRL, Bethesda, MD) containing 15% fetal calf serum (Gibco BRL) and 1% penicillin/ 'To whom correspondence should be addressed. Tel: +32 2 6293529; Fax: +32 2 6293408; Email: © UK Environmental Mutagen Society/Oxford University Press 1998 193 The human in vitro cytokinesis-blocked micronudeus (MN) assay has been extensively used for detection of clastogenic and aneugenic agents. In this test binucleate cells are generally considered to be the main target cell population for assessing genotoxic effect and almost no attention is paid to the biological information contained in mononucleate cells. In this study we analysed the frequencies of micronucleate mononucleates in a control population and after in vitro exposure to clastogens or aneugens. A clear increase in MN hi mononucleates was found only after exposure to aneugenic compounds. By means of fluorescence in situ hybridization using a chromosome 1-specific probe we further analysed the proportion of mononucleate cells with and without MN which were tetrasomic (tetraploid) and would have been induced during aneugen treatment by mitotic slippage. The data indicate that treatment with nocodazole induces tetrasomy for chromosome 1 (tetraploidy) and an increase in MN frequency in mononucleate diploid and tetraploid lymphocytes. The results thus confirm that some mononucleates pass mitosis without chromatid segregation to daughter nuclei. These data suggest that MN in mononucleates may be useful to distinguish clastogens from aneugens and increase the sensitivity of the test treatment with spindle poisons and progressed with or without separation of chromatids to the next cell cycle. Considering control of the separation of sister chromatids at the metaphase/anaphase transition, it was suggested by Hollo way et al. (1993) that some unknown protein involved in linkage of sister chromatids must be destroyed by the same machinery that proteolyses cyclin B. Recent data, moreover, indicate (Murray, 1995; Udvardy, 1996) that it probably takes place in three consecutive steps through protein ubiquitination. This implies that cyclin B destruction is required for chromosome decondensation, nuclear envelope reformation and cytokinesis, but not for chromatid separation. There is thus no reason to consider that cytochalasin B would interfere with chromatid separation in primary cells from normal individuals. In the presence of spindle poisons one may thus expect that some mononucleate cells in lymphocytes blocked by cytochalasin B may undergo chromatid separation, become polyploid (4N) and contain 4C or 8C DNA, depending on whether or not they are able to cross the Gl/S checkpoint of the next interphase. If no chromatid separation occurred the chromosomes should obviously show endoreduplication (2N, 4C or 8C). During recent years our laboratory has performed several studies with the cytochalasin B methodology comparing MN frequencies and characteristics in human lymphocytes exposed in vitro (Van Hummelen and Kirsch-Volders, 1992; Elhajouji et al, 1994, 1995, 1997; Kirsch-Volders et al, 1996; Tafazoli et al, 1995, 1996) or in vivo (Van Hummelen et al, 1993, 1994) to mutagens. From these data the following information is presented in this work: (i) background frequencies of MN in mononucleate and binucleate cells in a control population (non-exposed to known mutagens); (ii) frequencies of MN in mononucleate cells obtained after in vitro exposure to clastogens or aneugens; (iii) ploidy level of mononucleate cells after in vitro exposure to nocodazole. A.ElhnJo*Ajl, M.Cunha and M.Kirsch-VoWers Background frequency of MN-Mononucleates Qrmph A 2-3 3--4 4--S S--6 6-7 Range of MN-Mononndeatea (%o) Background frequency of bfN-binticleatea B 50 • 45 •40 •*36 -r 25 j20 15 10 5 0 0--1 Jill 1--2 2--3 3--4 4--5 S--6 6--7 Range of KQf-Mnucleatea (%o) Fig. 1. Percentage of individuals with a given spontaneous MN mononucleate frequency (A) and background frequencies of MN binucleates (B). All 240 healthy donors (230 men, 10 women) were included in this distribution. streptomycin (5000 IU/ml and 5000 |ig/ml; Life Technologies, Paisley, UK) and incubated in 5% CO2 in a humidified incubator at 37°C. The lymphocytes were stimulated with 2% phytohaemaggluUnin (PHA; Wellcome Diagnostics, UK) and treated with cytochalasin B (6 Hg/ml) at 44 h. After 72 h cultures were harvested. Cells were (...truncated)