abstract
The Karyotype stability in humans and animals relies on the coordinated function of the mitotic and meiotic cell division resulting in the accurate distribution of genetic material to the progenitor cells. These processes involve a series of inter-related cell organelles and activities such as the synthesis and function of the proteins in the spindle apparatus. Disturbances of the spindle apparatus may lead to aberrations in chromosome segregation and the production of cells that could contain a multiple of the complete haploid chromosome content (polyploidy) or deviations from the normal number of specific chromosomes (aneuploidy). Aneuploidy makes a significant contribution to both somatic and inherited diseases like infertility and cancer.
In contrast to DNA-reactiveagents, mutagens that induce their genotoxic effects through non-DNA reactive mechanism (e.g. aneugens) are expected to show thresholded dose reponse relationships. At the present time, the strongest data establishing a threshold for aneugens, was obtained only in vitro in human lymphocytes (Elhajouji et al., 1995; 1997). Determination of such thresholds in vitro cannot be used for risk evaluation in humans. It is not known how concentrations found in vitro in a particular type of cells, e.g. lymphocytes, can be extrapolated to somatic and/or germ cells in vivoin experimental animal models.Determination of potential thresholds in vivo that take into account the bioavailability/ADME (administration, distribution, metabolism, excretion) is of great importance for the assessment of risk in humans treated with such classes of drugs or environmental hazards.
The aim ofthe presented project was the determination of extensive dose-response relationships for the induction of micronuclei in vivoby the tubulin interacting aneugens colchicine, vinblastine, vincristineusing flow cytometric analysis in combination with fluorescence in situ hybridization with all centromere specific DNA probes. The comparative evaluation of the peripheral blood flow cytometry micronucleus test in Wistar Han rat and CD1 mouse exposed vinblastine, vincristine and colchicine after single dose applicationssuggest that spleen can play an important role in elimination of aneugen induced micronucleated reticulocytes in rat peripheral blood after single treatment. Therefore there is an advantage of using mouse peripheral blood for flow cytometry micronucleus test following single treatment protocol which is required for the detection of thresholds for aneuploidy induction in vivo.
Based on the flow cytometry analysis, the dose-response curve for the micronucleus induction for the all tested aneugens showed a non-linear dose-dependent increase in the micronuclei frequencies. To determine whether the micronucleus induction at higher doses was due to chromosome loss (aneuploidy) or chromosome breakage (clastogenicity), flow sorting of the micronucleated reticulocytes and further fluorescent in situ hybridization (FISH) using a mouse pan centromeric probe were performed. The flow cytometry and FISH data were combined and the statistical evaluation was performed to determine the threshold levels for chromosome loss in vivo. Based on mathematical modeling the threshold concentration for micronucleus induction with vinblastine, vincristine and colchicine was found at 0.35 mg/kg, 0.017 mg/kg and 0.49 mg/kg, respectively. In conclusion, flow cytometry in combination with thefluorescent in situ hybridization are reliable methods for aneuploidy assessment in vivo and the presented work clearly indicates the presence of thresholds for the investigated compounds in the peripheral blood in mice in vivo.