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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 thePig-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.

genes and environment

The Rat Pig-a Mutation Assay in Single and 28 Day-repeated Dose Study of Cyclophosphamide: The PIGRET Assay Can Detect the In Vivo Mutagenicity Earlier than the RBC Pig-a Assay