Evaluation Of Micronuclei Research Articles

Comparative mouse micronucleus evaluation in bone marrow and spleen using immunofluorescence and Wright's Giemsa

Bone marrow and spleen toxicity, clastogenicity and aneugenicity were analyzed in the CD 1 mouse using an antikinetochore antibody (AKA) procedure (Krishna et al., Mutation Res., 282, 159–169, 1992). Further, to verify the fluorescence micronucleus (MN) analysis, additional slides were stained with Wright's Giemsa and results were compared. 5 mice per sex were treated with cyclophosphamide (CP) (40 mg/kg) or vincristine (VC) (0.1 or 0.2 mg/kg). Slides were prepared 24 h postdose using a column fractionation procedure. Per animal, 400 total erythrocytes (TEs) for toxicity and 2000 polychromatic erythrocytes (PCEs) for MN per tissue were analyzed. In the fluorescent method, the clastogen, CP, produced MNPCEs predominantly devoid of kinetochores (K) and the aneugen, VC, produced mostly MNPCEs containing K. The MNP CE frequency did not differ significantly between tissues; however, it differed statistically between sexes. On an overall basis, spleen had significantly lower PCE to TE ratios compared to bone marrow. In general, CP and VC caused a small, but statistically significant decrease in PCE frequencies compared to controls, suggesting possible toxicity to these tissues at the given doses. The data on Wright's stain indicated that the proportion of PCEs and MNPCEs in general, were comparable to those using fluorescent stain. This study further confirms the usefulness of an AKA-staining technique in a multiple genetic endpoint evaluation under a single set of microscope conditions.

Micronucleus evaluation in peripheral blood reticulocytes of mice treated with procarbazine hydrochloride or mitomycin C

The usefulness of the acridine orange (AO) supravital staining technique for the mouse peripheral blood reticulocyte micronucleus test was investigated independently by three laboratories using the known clastogens procarbazine hydrochloride (PCZ) and mitomycin C (MMC). In all three laboratories the highest frequencies of micronucleated peripheral blood reticulocytes were observed 48 h after treatment of mice with a single dose of either MMC or PCZ. The animals responded to both chemicals in a dose-dependent manner. Although similar qualitative results were observed, mean micronucleus frequencies induced by a particular dose of a given test chemical did vary quantitatively among the three laboratories. This was most probably due to the use of slightly different scoring criteria by each examiner. This aspect needs special attention. To minimize inter-laboratory variability, therefore, we recommend establishing unequivocal criteria to distinguish the subclasses of reticulocytes. These should then be used consistently by all investigators using this method. The most striking advantages of the AO supravital staining technique were the ease of slide preparation, the ease with which reticulocytes and mature erythrocytes could be distinguished by the examiners, and the occurrence of numerous scorable reticulocytes in each microscopic field, which greatly speeded up the manual counting process. The disadvantages of the staining technique were the limited scoring time due to the rapid fading of the fluorescence stain, the degradation of the cells with time, and the frequent need to search for adequate scoring areas within a microscopic field. Based on the data of this study the authors conclude that the AO supravital staining technique is highly suitable for the micronucleus assay in erythrocytic cells of mouse peripheral blood. In addition, we consider the mouse peripheral blood reticulocyte micronucleus test to be a useful tool with which to investigate the dastogenic potential of chemicals in vivo. As pretreatment of mice with Aroclor 1254 markedly increased the effect of PCZ on micronucleus induction, we suggest that the inclusion of inducers of drug metabolizing enzymes in the micronucleus test would be useful for tile detection of the clastogenic potential of promutagenic chemicals.

Use of the cytokinesis-block micronucleus method in mouse splenocytes

Mouse splenocytes have been used in the cytokinesis-block method for the evaluation of micronuclei induced by mutagenic agents in vitro as well as in vivo. Stimulation with concanavalin A for 48 h followed by 16–24-h treatment with 5 μ/ml cytochalasin B was found to be an optimum condition to obtain micronuclei in the binucleated splenocytes after the cells were cultured in vitro. Under the above conditions splenocytes from mice pretreated with a single i.p. injection of cyclophosphamide gave a significant increase in micronucleus production. This increase was dependent on the dose of cyclophosphamide ( r = 0.99). A dose of 50 mg/kg resulted in 22% of the binucleated cells producing micronuclei, more than 20 times the level in the untreated control. The increase was also dependent on the time of cyclophosphamide injection before removal of the spleen. A duration of 4–8 h after cyclophosphamide injection gave rather sharp optimum values for the production of micronuclei. When splenocytes from non-treated mice were treated with mitomycin C together with cytochalasin B in the above in vitro condition, there was a significant increase in micronucleus production in the binucleated cells. It was also dependent on the dose of mitomycin C ( r = 0.975) and a dose of 0.5 μg/ml resulted in a more than 20-fold increase over the untreated control. Thus, the use of mouse splenocytes in the cytokinesis-block micronucleus assay was shown to be sensitive enough for testing mutagenic agents in vivo as well as in vitro.

Micronucleated lymphocytes in people occupationally exposed to potential environmental contaminants: the age effect

This work is part of a research project on 2 groups of tannery workers (i.e., workers employed in the tanning process and those employed in the finishing department), and 2 control groups consisting of individuals paired with each exposed person according to sex, age and smoking habit. The whole study included the evaluation of micronuclei as well as of chromosomal aberrations and sister-chromatid exchanges in peripheral blood lymphocytes. Data on micronucleus analysis in both controls and exposed persons are shown in this paper. There was no statistically significant difference between MN frequencies in the 2 groups of exposed and controls, nor any positive correlation with smoking habit. The effect of age on basal frequency of micronucleated cells clearly emerges in the present study: both controls and exposed show an increase in MN frequency due to age. This could be correlated with a higher sensitivity to breaks, rearrangements or aneuploidogenic events of circulating lymphocytes in aged people.