Indication for thresholds of chromosome non-disjunction versus chromosome lagging induced by spindle inhibitors in vitro in human lymphocytes

Mutagenesis vol.12 no.3 pp.133-140, 1997 Indication for thresholds of chromosome non-disjunction versus chromosome lagging induced by spindle inhibitors in vitro in human lymphocytes Azeddine Elhajouji1'3, Fabian Tibaldi2 and Micheline Kirsch-Volders1 'Anthropogenetics Laboratory, Vrije Universiteit Brussel, Pleinlaan 2, 1050-Brussels, Belgium and Institute de Calculo, University of Buenos Aires, Argentina 3 To whom correspondence should be addressed Introduction Aneuploidy characterizes any cell or organism with a chromosome number which deviates from a multiple of the haploid • UK Environmental Mutagen Society/Oxford University Press 1997 133 Risk assessment from exposure to spindle inhibitors should take into account the possibility of threshold concentrationresponse curves for aneuploidy induction. We analysed concentration-dependent induction of chromosome nondisjunction by well known spindle poisons (colchicine, carbendazim, mebendazole and nocodazole) and a reference clastogen, methyl methanesulphonate (MMS) in vitro in human lymphocytes; and integrated these findings with earlier results of chromosome loss in micronuclei. Chromosome non-disjunction was estimated on cytokinesis-blocked lymphocytes after simultaneous fluorescent in situ hybridization labelling with two chromosome-specific centromeric probes (chromosomes 1 and 17). The frequencies of spontaneous non-disjunction showed important inter-individual variations and were surprisingly high (7.04-15.39%). Lower concentrations of aneugens did not induce a statistically significant increase of non-disjunction frequencies over the respective control levels, whereas higher concentrations clearly induced a concentration-dependent increase in the non-disjunction frequencies with the four aneugens tested. On the contrary, even at high concentrations, MMS induced a slight increase in the frequency of non-disjunction but without being statistically significant when compared with the control frequencies. We estimated the inflection points, the first statistically significant concentrations, the last nonstatistically significant concentrations and the number of events from concentration-response curves of chromosome non-disjunction and chromosome loss. A threshold-type of concentration-response for non-disjunction is highly probable for colchicine and nocodazole. For carbendazim and mebendazole the inflection point fell above the first statistically significant concentrations. But since it is obvious from dose-response curves where the inflection point/ threshold lies, it appears that the model might be picking up some irregularities (possibly due to experimental variability in the dose—response curve at concentrations greater than the threshold). For accurate estimation of the threshold, analysis of more concentrations or more cells might be needed. Our data strongly indicate that in cultured human lymphocytes chromosome non-disjunction is a major mechanism of aneuploidy induction by spindle inhibitors and since non-disjunction occurs at lower concentration than chromosome loss, the aneuploidy threshold should be estimated on the basis of non-disjunction rather than on micronuclei frequencies (chromosome loss). set of chromosomes. Several potential targets/mechanisms for chemically-induced aneuploidy are known (Liang et ai, 1985; Dellarco et ai, 1986). Theoretically, these include: the chromosomes themselves (particularly the centromeres and telomeres), the kinetochore proteins, microtubule synthesis and assembly, the formation of mitotic dividing spindle, the synthesis and functioning of the polar bodies, the movement of the segregating chromosomes on the spindle, membrane modifications and those meiosis-specific events such as formation of the synaptonemal complexes and recombinational exchanges. Interference with such targets can lead to either chromosome loss, resulting from lagging chromosomes at the anaphase, or chromosome non-disjunction, where both sister chromatids migrate to the same daughter nucleus. The incidence of chromosomal non-disjunction in man is relatively high compared with that in rodents (Bond and Chandley, 1983). Meiotic non-disjunction has been implicated in a wide range of abnormalities, such as fetal wastage, prenatal and infant mortality, congenital malformation and mental retardation (Bond and Chandley, 1983; Hook, 1985; Hoffmann et ai, 1986). Mitotic non-disjunction is closely associated with malignant diseases and neoplastic transformations (Evans, 1985). Indeed, there is considerable evidence that chemical exposure leads to the induction of specific aneuploidies and that they have a role in tumour progression (Oshimura and Barrett, 1986; Fearon and Vogelstein, 1990; Herens et ai, 1992; Haesen et ai, 1993; Van Goethem et ai, 1995). When comparing the type of mutations, ranging from gene mutations on a single base (point mutation) to structural or numerical changes of whole chromosomes with a number of targets possibly involved in their induction, it is clear that probably only genome mutations (numerical chromosome changes) result from multi-target interaction with the mutagen; indeed chromosome non-disjunction and loss are the consequences of the binding of aneugens on spindle or centriolar tubulins, scaffolding and nuclear proteins or centromere and kinetochore regions. From a theoretical point of view, it is considered that mutagens which can induce a lesion after interaction with a single target, e.g. covalent binding with DNA, show doseresponse curves without threshold; indeed potentially any DNA lesion which is not repaired may lead to a mutation. Alternatively, mutations which require the involvement of more than one target should follow threshold dose curves (Crebelli and Carere, 1993; Parry et ai, 1993). As a consequence, for those mutagens which could be proven to exert their effects only above a given threshold concentration, risk assessment should be guided by this information. Up to now, however, only few data are available to sustain these concepts (Parry et ai, 1994). Therefore, our aim was to study in vitro in human lymphocytes the concentration curve responses of model multi-target acting aneugens with the most sensitive cytogenetic methodologies allowing the detection of mutagenic effects at low concentrations and to define the most appropriate mathematical models to identify the critical concentrations. A.Elhajouji, F.Tibaldi and M.Kirsch-Volders Materials and methods Chemicals MBC (CAS: 10605-21-7) was purchased from Aldnch Chemie (Steinheim, Germany); MEB (CAS: 31431-39-7) from Sigma Chemical Co., Brussels, Belgium; NOC (CAS: 31430-18-9) was provided by Janssen Pharmaceutica, Beerse, Belgium. These three chemicals were dissolved in dimethylsulphoxide (DMSO; Merck, Darmstadt, Germany) for spectroscopy. Methyl methanesulphonate (MMS; CAS: 66-27-3) was purchased from Merck, and dissolved in phosphate-buffered saline (PBS). COL (CAS: 64-86-8) was purchased (...truncated)