-The Pig-a gene mutation assay using perpherial blood erythrocytes is being investigated as a screening tool for assessing mutagenicity in vivo. In this study, we evaluated two distinct approaches for performing the Pig-a assay in rats. We used antibodies to CD45 or the erythroid marker HIS49 to identify red blood cells (RBCs), and then monitored the kinetics of Pig-a mutant frequency, as measured by the frequency of CD59-deficient RBCs, in rats treated with the genotoxic chemicals, N-ethyl-N-nitrosourea, cyclophosphamide, 4-nitroquinoline-1-oxide, and ethylmethanesulfonate. In some instances, micronucleus frequency also was measured in the same animals. Time-and dose-related increases in Pig-a mutant frequency were found in all the chemical-treated groups, except for the groups treated with cyclophosphamide, which was a potent inducer of micronuclei. The two different approaches we employed were comparable for measuring induced mutant frequencies, but our historical data showed that the mean background frequencies for the CD45/CD59 method and the HIS49/CD59 method were 12.7 × 10 -6 and 5.5 ×10 -6 , respectively. The relatively low, stable background mutant frequency associated with the HIS49/CD59 method indicates that it may have greater power for discriminating weak induced responses. These results suggest that the HIS49/CD59 method is a promising tool for measuring Pig-a mutant RBCs. In addition, differences in their manifestation kinetics and in their relative sensitivity for detecting different test compounds suggest that the combination of the Pig-a assay and the micronucleus assay may be effective in identifying in vivo genotoxicity.

The peripheral blood Pig-a assay is now recognized as one of genetic toxicology test to detect the in vivo mutagenic potential of chemical. A previous report on interlaboratry trial by Japanese research group has shown that the rat Pig-a assay with an antibody binds to an erythroid marker is transferable and reproducible. By using this approach, we evaluated the capability of the Pig-a assay protocol to integrate into the general toxicity studies (single or repeated dose study). Both Pig-a assay in total red blood cells (RBC Pig-a assay) and Pig-a assay in reticulocytes (PIGRET assay) were performed before and at days 8, 15 and 29 following single or 28-daily treatments of cyclophosphamide (CP). The difference in the kinetics of increase in Pig-a mutant frequency (MF) between total red blood cell (RBC) and reticulocyte (RET) was found in the single dose study; RET Pig-a MF was temporary increased at days 8 and 15, while RBC Pig-a MF was increased only at day 15. In the repeated dose study, the RET Pig-a MF was increased in the high dose group at day 29, though it was the result under the conditional statistical analysis which excluded one outlier in the control group. The manuscript by Dertinger et al, also showed the increase of Pig-a MFs in both RBCs and RETs, suggesting that the Piga assay for the repeated dose study is feasible to detect the mutagenicity of CP. Taken together, the increase of Pig-a MF was detectable under the both single and 28-day repeated dose study with CP. These results suggest that the Pig-a assay approaches are practical in the general toxicity studies. In addition, the PIGRET assay is an advantageous method at the point that the increase in mutant cells is more detectable at an early stage compared with the RBC Pig-a assay. It is thought that this phenomenon is based on the differentiation stage of an erythroid lineage.