DNA content determination of micronucleated polychromatic erythrocytes induced by clastogens and spindle poisons in mouse bone marrow and peripheral blood

Abstract

The frequencies and DNA distributions of micronuclei (MN) in polychromatic erthrocytes (PCEs) from bone marrow (BM) and peripheral blood (PB) of mice after four different treatments were determined by flow cytometry. PCEs were differentiated from normochromatic erythrocytes (NCEs) using the fluorescent RNA stain Thiazole orange, while MN in erythrocytes were detected with the DNA stain Hoechst 33342. The treatments were X-irradiation (1 Gy), cyclophosphamide (CPA; 30 mg/kg), vincristine sulphate (VCR; 0.08 mg/kg), and cholchicine (COL; 1 mg/kg). All treatments showed increased frequencies of micronucleated PCEs at 30 h after treatment in BM and at 50 h in PB. The clastogens (X-irradiation and CPA) and the spindle poisons (VCR and COL) could be grouped according to the fluorescent characteristics of the induced MN as well as the relative frequency of small (0.5-2% of the diploid DNA content) and large (2-10%) MN. In PB the relative frequency of large MN was lower than in BM, indicating that they were partly eliminated before entrance into the peripheral circulation.

Flow cytometric analysis of micronucleus induction in rat bone marrow polychromatic erythrocytes by 1,2;3,4-diepoxybutane, 3,4-epoxy-1-butene, and 1,2-epoxybutane-3,4-diol

“…The HO342-TO dot plots in Figure 1 are very similar to those obtained from previous studies of MPCE in mouse bone marrow, with populations of PCEs, NCEs, MPCEs, and nucleated bone marrow cells (17). The HO342-TO dot plots in Figure 1 are very similar to those obtained from previous studies of MPCE in mouse bone marrow, with populations of PCEs, NCEs, MPCEs, and nucleated bone marrow cells (17).…”

“…A threshold level was established for TO fluorescence, which excluded virtually all NCEs (Figure 1b,c) (17). A threshold level was established for TO fluorescence, which excluded virtually all NCEs (Figure 1b,c) (17).…”

“…The procedure for sample collection and purification is essentially identical to that described for mouse bone marrow (14,17). One femur was excised, and the bone marrow was flushed out into Falcon 2052 tubes (Becton Dickinson, Lincoln Park, NJ) with a syringe filled with 4 ml of RPMI-1640 medium (HyClone, Cramligton, UK).…”

“…The fixing and staining steps are identical to those described for mouse bone marrow cells (17). The cells were fixed in a freshly made, 1% solution of glutaraldehyde (Merck, Darmstadt, Germany) in PBS with 30 µg/ml of sodium dodecyl sulphate (Fluka Chemie, Buchs, Switzerland) added to sphere the erythrocytes (18).…”

“…Various techniques have been applied in the mouse, the most common species in the micronucleus test (15,18,22,30,34). This technique has also been applied successfully to analyse MN in mouse bone marrow PCE (14,17). This dose level is the lowest detected by any method for biological dosimetry using cytogenetic damage as an endpoint under controlled conditions.…”