Micronuclei induced by aneugens and clastogens in mononucleate and binucleate cells using the cytokinesis block assay

Christiane Rosefort 0 Evelyne Fauth 0 Heinrich Zankl 0 0 Department of Human Biology and Human Genetics, University of Kaiserslautern D-67663 Kaiserslautern , Germany The human in vitro micronucleus (MN) test has become a fast and reliable assay for mutagenicity testing. Currently, this assay is mostly performed with cytochalasin B, which prevents cytokinesis, resulting in polynucleated cells. The number of nuclei per cell indicates the number of nuclear divisions that have occurred since the addition of cytochalasin B. It is recommended that MN are only counted in binucleated lymphocytes, because these cells have finished one nuclear division. Therefore, almost no attention has been paid to MN in mononucleated cells. However, recent studies have indicated that aneugens, but not clastogens, also induce MN in mononucleates. In order to evaluate mononucleates to distinguish between aneugenic and clastogenic effects, we tested some typical aneugens and clastogens in whole blood lymphocyte cultures of four donors with the cytokinesis block micronucleus (CBMN) assay. Results showed that the aneugens diethylstilbestrol (80 mM), griseofulvin (25 mg/ml) and vincristine sulphate (15 mg/ml) increased MN frequencies in mononucleated and binucleated cells, whilst the clastogens mitomycin C (500 ng/ml), bleomycin (6 mg/ml) and doxorubicin (20 mg/ml) increased MN frequency only in binucleates. We also tested the Y heterochromatin decondensing drug berenil (300 mg/ml). Berenil induced an extremely high number of MN in mononucleated as well as in binucleated cells, indicating an aneugenic action. This was confirmed by centromere labelling. The results suggest that MN in mononucleates may be an interesting additional parameter in the CBMN assay. Future studies should clarify whether the micronucleated mononucleate cells have escaped the cytokinesis block and become polyploid. Introduction For the purposes of mutagenicity testing metaphase chromosomes are routinely screened for numerical and structural chromosome aberrations (CA) to detect damage to DNA and cell division. However, metaphase analysis is very time consuming and needs highly skilled personnel. Therefore, the micronucleus (MN) assay was developed as a short-term screening test. In this method, CA are detected indirectly via chromatin loss from the nucleus leading to MN in the cytoplasm of the cell. MN are defined as small, round, DNAcontaining cytoplasmic bodies formed during cell division by loss of both acentric chromatin fragments and whole chromosomes. This chromatin loss can be induced by different mutagens in vivo and in vitro. Thus, the in vitro MN assay has become a fast and reliable test system for detecting mutagenic action (for reviews see Fenech, 2000; Kirsch-Volders et al., 2002). Because cell division is necessary for the generation of MN, it is recommended that MN are scored by the cytokinesis block micronucleus (CBMN) assay developed by Fenech and Morley (1985a,b). In the CBMN assay cultures are treated with cytochalasin B, which is an inhibitor of actin polymerization. Cytochalasin B prevents cytokinesis but not nuclear division, resulting in cells with multiple nuclei (Carter, 1967). Using this method it is possible to identify cells which have divided once, because they show two nuclei. Originally, the CBMN test focused exclusively on such binucleate cells (Fenech and Morley, 1985a,b). However, it was suggested that MN in mononucleate cells could provide complementary information, because mononucleated cells should indicate damage present in vivo before the start of cell culture, making these cells interesting for biomonitoring purposes (Kirsch-Volders and Fenech, 2001). As far as we know, Elhajouji and colleagues (1998) were the first to screen for MN induction in mononucleate cells using the CBMN assay. In the past, several attempts have been made to distinguish between the aneugenic and clastogenic action of test compounds. The first approach was based on measuring the diameter of MN. It was assumed that the loss of whole chromosomes induced by aneugens would result in larger MN than those induced by clastogens, which generate only chromosomal fragments (Yamamoto and Kikuchi, 1980; Hoegstedt and Karlsson, 1985; Wakata and Sasaki, 1987; Tinwell and Ashby, 1991; Ferguson et al., 1993; Komae et al., 1999). This method seems to be applicable, but it is very time consuming and will be subject to difficulties in species with karyotypes that include chromosomes of very different size. Therefore, other authors used the measurement of MN DNA content (Heddle and Carrano, 1977; Nuesse and Kramer, 1984; Pincu et al., 1985) or combined three parameters, area of MN, C-band-positive material and DNA content of MN (Vanderkerken et al., 1989; Vanparys et al., 1990; Van Hummelen et al., 1992; Schneider et al., 1995). Currently, the most widespread and reliable assays identify whole chromosomes in MN by labelling their kinetochores or centromeres. The CREST assay detects kinetochore proteins by immunofluorescence techniques (labelled MN are termed K+; Degrassi and Tanzarella, 1988; Hennig et al., 1988; Thomson and Perry, 1988; for a review see Natarajan et al., 1996), while the FISH assay labels centromeric DNA sequences (labelled MN are termed C+; Becker et al., 1990; Migliore et al., 1993; Norppa et al., 1993; for a review see Natarajan et al., 1996). However, only a few laboratories routinely use these techniques (Surralles and Natarajan, 1997), because they are very costly. Elhajouji and colleagues (1998) reported a new and easy way to distinguish between aneugenic and clastogenic actions in the CBMN assay. The authors showed that some aneugens increased MN frequencies in mononucleate as well as binucleate cells, whilst clastogens induced MN solely in binucleates. This difference may allow discrimination between MN generated by aneugens and clastogens. To test this hypothesis, some well-known aneugens and clastogens were studied for their ability to induce MN in mononucleates. Materials and methods Donors and cell cultures Peripheral blood was obtained once from two female (25 and 24 years of age, referred to as donor 1 and 2, respectively) and two male (26 and 24 years of age, referred to as donor 3 and 4, respectively) unrelated donors who had normal karyotypes. For each donor, one series of cultures was prepared with two parallel cultures (duplicates) for every concentration of tested mutagen. We used whole blood cultures as recommended by Migliore et al. (1989). For each culture, 0.8 ml of heparinized blood were added to 8 ml of RPMI 1640 medium (supplemented with 300 mg/ml glutamine; Invitrogen) containing 15% foetal calf serum (Biochrom), 1% penicillin/streptomycin (Invitrogen) and 0.2 ml phytohaemagglutinin (22 mg/ml; Invitrogen). After 44 h cultivation, all cultures were supplemented with cytochalasin B (Serva). For the stock solution 1 mg cytochalasin B was dissolved in 0.25 ml of DMSO, resulting i (...truncated)