Further development of the rat Pig‐a mutation assay: Measuring rat Pig‐a mutant bone marrow erythroids and a high throughput assay for mutant peripheral blood reticulocytes

Abstract

Recent studies indicate that the Pig‐a assay is a promising tool for evaluating in vivo mutagenicity. We have developed novel rat Pig‐a assays that facilitate measuring mutant frequencies in two early arising populations of blood cells, bone marrow erythroids (BMEs) and peripheral blood (PB) reticulocytes (RETs). In these assays, bone marrow cells of erythroid origin and PB red blood cells (RBCs) were identified using an antibody against rat erythroid‐specific marker HIS49. In addition, RETs were selectivity enriched from PB using magnetic separation of cells positive for CD71, a transferrin receptor expressed on the surface of BMEs and RETs, but not on the surface of mature RBCs. With magnetic enrichment, more than 1 × 10⁶ CD71‐positive RETs could be evaluated by flow cytometry for Pig‐a mutant frequency within 5 to 8 min. CD59‐deficient RET and BME frequencies of more than 100 × 10⁻⁶ and 80 × 10⁻⁶ were detected 1 week after treating rats with 40 mg/kg N‐ethyl‐N‐nitrosourea; by comparison, the frequency of CD59‐deficient total RBCs in these rats was 13.2 × 10⁻⁶. The frequency of spontaneous Pig‐a mutant RETs and BMEs was less than 5 × 10⁻⁶ and 15 × 10⁻⁶, respectively. Since ∼98% of nucleated cells in the BME fraction were erythroblasts, it should be possible to use BMEs to determine the spectrum of CD59‐deficient Pig‐a mutations in cells of erythroid lineage. Conducting concurrent Pig‐a assays on RETs and BMEs may be useful for evaluating the in vivo mutagenicity of chemicals, especially when prolonged mutant manifestation is not feasible or when the confirmation of mutation induction is necessary. © Environ. Mol. Mutagen. 2011. Published 2011 Wiley‐Liss, Inc.