Comparison of separated erythrocyte preparations and manual smears of bone marrow in showing micronucleus induction by clastogens and aneuploidogens in mouse

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Abstract
The removal of nucleated cells from bone marrow cell suspensions by cellulose column separation and further purification in a Percoll gradient, coupled with slide preparation by a cytocentrifuge, produces uniform preparations of pure well-spread erythrocytes. The use of separated erythrocytes has been a considerable improvement in the in vivo micronucleus (MN) assay, because the optimal cell morphology and the possibility of scoring a high number of polychromatic erythrocytes (PCEs) in a short time are factors contributions to the sensitivity of the assay. As the separation procedure may selectively retain cells containing MN, a study comparing the performance of the erythrocyte fractionation technique and standard manual smear preparation was conducted in male NMRI mice treated with a single injection of two clastogens (cyclophosphamide, 25 or 50 mg/kg, or methylmethane sulfonate, 40 or 80 mg/kg) or two aneuploidogens (colchicine, 0. 5 or 1 mg/kg, or vincristine sulfate, 0. 05 or 0. 1 mg/kg). Both the preparation methods were clearly able to detect the MN-inducting and toxiceffects of the treatments. After treatment with cyclophophamide and methlmethane sulfonate, the frequency of micronucleated PCEs (MNPCEs) was, respectively, 1. 6 and 1. 8 times higher in the fractionated erthrocytes than in whole bone narrow. Morphological characterization of the MN indicated that this phenomenon was due to improved indentification of small micronuclei in the flat cytospun cells of the fractionated erythrocyte preparations. On the other hand, the purified erthrocyte slides contained only about half the number of MNPCEs observed in standard sm, ears, in samples collected from mice treated with colchicine. This finding may be due to the loss of some of the MNPCEs harbouring a large MN in a too-tightly packed cellulose column. The frequency of PCEs was consistently higher in the fractionated erythrocyte preparations, which indicates that the Percoll gradient step may require further refinement. In conclusion, the use of separated erythrocytes instead of manual smears appears to offer a possibility for improved detection of clastogens. On the other hand, the loss of normochromatic erythrocytes and of PCEs with large MN are problems which require the refinement of the separation techniques.

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