Manifestation of Pig-a mutant bone marrow erythroids and peripheral blood erythrocytes in mice treated with N-ethyl- N-nitrosourea: Direct sequencing of Pig-a cDNA from bone marrow cells negative for GPI-anchored protein expression

• Published in: Mutation research Vol. 723; no. 1; pp. 36 - 42
• Main Authors: Kimoto, Takafumi, Suzuki, Kumiko, Kobayashi, Xiao mei, Dobrovolsky, Vasily N., Heflich, Robert H., Miura, Daishiro, Kasahara, Yoshinori
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 14.07.2011; Elsevier; Elsevier BV
• Subjects: Animals; Antibodies; Biological and medical sciences; Bone Marrow Cells - drug effects; CD24; Erythrocytes; Erythrocytes - drug effects; Ethylnitrosourea - toxicity; Flow cytometry; Fundamental and applied biological sciences. Psychology; Gene expression; Gene mutation; Genetics of eukaryotes. Biological and molecular evolution; Glycosylphosphatidyl inositol; Glycosylphosphatidylinositols - metabolism; Medical sciences; Membrane Proteins - genetics; Mice; Mutagenicity Tests - methods; Mutation; Phosphatidylinositol glycan complementation class A gene; Protein synthesis; Rodents; Sorting; Time Factors; Toxicology; Phosphatidylinositol glycan complementation class A gene; Flow cytometry; Glycosylphosphatidyl inositol; CD24; Gene mutation; Sorting; Erythroid cell; Nitrosoureas; Blood; Phosphatidylinositol; Gene; Bone marrow; Complementation; Sequencing; Ungulata; Inositol; Rodentia; Glycan; Protein; Pig; Vertebrata; Mammalia; Complementary DNA; Mouse; Phosphatidylinositol glycan; complementation class A gene; Artiodactyla; Mutation
• ISSN: 1383-5718; 0027-5107; 1879-3592
• DOI: 10.1016/j.mrgentox.2011.03.016

► We developed a flow cytometric Pig-a assay for measuring gene mutation in the mouse. ► We used this assay to elucidate the generation of mutations in the Pig-a assay. ► The kinetics of Pig-a mutant manifestation is more rapid in bone marrow than in peripheral blood. ► CD24-negative erythroids have mutation in the Pig-a gene. ► The Pig-a assay on red blood cells is useful for the detection of in vivo mutagenicity. Our previous rat studies indicate that the endogenous Pig-a gene is a promising reporter of in vivo mutation and potentially useful as the basis for an in vivo genotoxicity assay. The function of the Pig-a protein in the synthesis of glycosylphosphatidyl inositol (GPI) anchors is conserved in variety of eukaryotic cells, including human and rodent cells, which implies that Pig-a mutants can be measured in a similar manner in different mammalian species. In the present study, we developed a flow cytometric Pig-a assay for rapidly measuring gene mutation in the mouse. An antibody to TER-119, a specific cell-surface marker of murine erythroid lineage, was used to identify erythrocytes in peripheral blood (PB) and erythroids in bone marrow (BM). An antibody to CD24, a GPI-anchored protein, was used to identify Pig-a mutants as CD24-negative cells. CD-1 mice were administered a single dose of 100 mg/kg N-ethyl- N-nitrosourea (ENU), and PB and BM were collected at 1, 2, and 4 weeks after dosing. While the Pig-a mutant frequency (MF) in PB was increased moderately at 2 and 4 weeks after ENU dosing, the Pig-a MF in BM was strongly increased starting at 1 week after the dosing, with the elevated MF persisting for at least 4 weeks after the dosing. We also used flow cytometric sorting to isolate CD24-negative erythroids from the BM of ENU-treated mice. cDNA sequencing indicated that these cells have mutations in the Pig-a gene, with base-pair substitutions typical of ENU-induced mutation spectra. The results indicate that the Pig-a mutation assay can be adapted for measuring mutation in BM erythroids and PB of mice. Taken together, the data suggest that Pig-a mutants are fixed in the BM, where they further proliferate and differentiate; erythrocytes derived from these BM Pig-a mutants transit from the BM and accumulate in PB.