The optimized conditions for the in vitro micronucleus (MN) test procedures using chamber slides

Optimized conditions for an in vitro micronucleus (MN) test procedure were defined using a chamber slide that enabled the preparation of fine specimens without undergoing complicating procedures using culture dishes. The issues investigated are 1) the effect of slide materials on the adhesion of cells, 2) the number of seeding cells necessary to obtain an adequate number of cells for observation and 3) effects of hypotonic treatment and fixation on the cytoplasmic: nuclear area ratio. In addition, we determined cell viability in each chamber using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. The results of the investigation were as follows: 1) cell adhesion was best using plastic slides, 2) the optimum number of cells for seeding was 6.6×103 cells/cm2, 3) the best condition for hypotonic treatment was incubation in 75 mM KCl at 37°C for 5 min, and 4) the best condition for fixation was treatment of cells twice for about 2 min in icecold methanol containing 6% acetic acid. Finally, the result of the MTT assay correlated with the number of viable cells in chamber as determined by the trypan blue dye exclusion assay.An in vitro MN test was conducted under these conditions using the known clastogens, Mitomycin C and dimethylnitrosamine. These clastogens dose-dependently induced a significant increase in the number of micronucleated cells with positive responses at concentrations approximately 10 times lower than those of the chromosomal aberration test. On the other hand, the frequency of micronucleated cells in the solvent control was stable and low (0.4-1.7%). These results indicated that the in vitro MN test has a high level of sensitivity to clastogens.It was concluded that the in vitro MN test using chamber slides is a rapid, simple and sensitive method to detect clastogens.