THE USE OF FLUORESCENCE IN-SITU HYBRIDIZATION FOR THE DETECTION OF ANEUGENS IN CYTOKINESIS-BLOCKED MOUSE SPLENOCYTES

In the mouse splenocyte assay cytokinesis-blocked micronuclei (MN) and fluorescent in situ hybridization techniques were applied to study aneuploidy following in vitro/in vivo treatments. MN can be formed by lagging acentric chromosome fragments or whole chromosomes. In order to discriminate MN produced by agents causing chromosome breakage (clastogens) from those arising following treatment with agents causing spindle malfunctioning (aneugens) the mouse centromere satellite DNA probe was used in combination with the fluorescent in situ hybridization technique. MN in mouse splenocytes were induced in vitro by colchicine, vincristine sulphate and vinblastine. For in vivo treatment, mice were injected intraperitoneally with diethylstilbestrol (DES) and hexamethylphosphoramide (HMPA) or whole body X-irradiated. Depending on the presence of the fluorescent signal in the MN following in situ hybridization with centromere-specific probe MN in the binucleated splenocytes were scored as centromere-positive (C +) or centromere-negative (C -). Treatment of mouse splenocytes (in vitro) with potent aneugens such as colchicine, vincristine and vinblastine increased significantly the number of centromere-positive MN (P < 0.001). Following in vivo treatment and in vitro culturing of mouse splenocytes significant (P < 0.001) aneugenic activities were observed with indirectly-acting chemicals such as DES and HMPA. X-irradiation caused a slight increase in the frequency of centromere-positive MN (approximately 20%) in binucleated mouse splenocytes. In addition the Giemsa C-banding technique was used to detect C + MN and a comparison was made between the mouse centromere specific probe and the conventional C-banding technique to detect centromeres in MN.