The in vivo Pig-a mutation assay has been adapted for measuring mutation in rats, mice, monkeys, and humans. To date, the assay has been used mainly to assess the mutagenicity of chemicals that are known to be powerful point mutagens. The assay has not been used to measure the biological effects associated with ionizing radiation. In this study, we modified the Pig-a gene mutation assay (Kimoto et al. [2011b]: Mutat Res 723:36-42) and used 3-color staining with fluorescently labeled anti-CD24, anti-TER-119, and anti-CD71 to detect the Pig-a mutant frequencies in total red blood cells (RBCs) and in reticulocytes (RETs) from X-irradiated mice. Single exposures to X-irradiation resulted in dose- and time-dependent increases in Pig-a mutant frequencies, and these subsequently declined over time returning to background frequencies. The same total amount of radiation, delivered either as a single dose or as four repeat doses at weekly intervals, increased Pig-a mutant frequencies to comparable levels, reaching maxima 2-3 weeks after the single dose or 2-3 weeks after the last of the repeat doses. These increased frequencies subsequently returned to background levels. Our results indicated that the 3-color Pig-a assay was useful for evaluating the in vivo genotoxicity of radiation.
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