The fetal blood erythrocyte micronucleus assay: classification of RNA-positive erythrocytes into two age populations by RNA aggregation state.

The use of a fluorescent stain containing Hoechst 33258 and pyronin Y in the fetal mouse transplacental micronucleus assay allows classification of erythrocytes into three subpopulations on the basis of RNA staining, and permits micronuclei to be scored in all three subpopulations. The youngest erythrocytes stain uniformly positive for RNA (UEs). In older erythrocytes RNA aggregates to give the cells a stippled appearance (SEs) and ultimately disappears, leaving cells which do not stain positively for RNA. Frequencies of micronucleated UEs and SEs were determined at 30 and 48 h following a single dose of methyl methanesulfonate, benzo[a]pyrene, lasiocarpine, monocrotaline or heliotrine. With each agent and dose tested, the frequency of micronuclei increased first in the younger UEs and later in SEs. The use of the Hoechst/pyronin staining procedure, which permits DNA to be distinguished from RNA, minimizes the potential for mis-scoring RNA artefacts as micronuclei and also increases the efficiency of the assay by permitting two age populations of erythrocytes to be scored in each sample.