Pig-a mutations in bone marrow erythroblasts of rats treated with 7,12-dimethyl-benz[a]anthracene.

Flow cytometry-based phenotypic detection of red blood cells (RBCs) deficient in surface markers anchored by glycosylphosphatidylinositol (GPI) is an efficient tool for monitoring somatic mutation in mammalian species. Biochemical considerations suggest that GPI-anchored marker-deficient RBCs found in peripheral blood are due to mutations in the endogenous X-linked phosphatidylinositolglycan, class A gene (Pig-a gene). Yet the linkage between the detected mutant phenotype and the actual mutation in the Pig-a gene is difficult to establish directly in mammalian RBCs that are naturally free of genomic DNA and may have only traces of heavily degraded mRNA. We have traced the origin of the marker-deficient RBC phenotype in the precursors of peripheral RBCs, bone marrow erythroid cells (BMEs, also known as erythroblasts), in rats treated by gavage with 75 mg/kg of the potent mutagen, 7,12-dimethyl-benz[a]anthracene (DMBA). The frequencies of marker-deficient BMEs were significantly increased in DMBA-treated rats. We identified Pig-a mutations in sorted mutant phenotype BMEs. The spectrum of DMBA-induced Pig-a mutations in erythroid lineage cells was identical to the spectra of mutations previously determined for the Pig-a and for another X-linked reporter gene, hypoxanthine-guanine phosphoribosyltransferase gene, in cells of lymphoid lineage, spleen T-lymphocytes. Our observations lend additional support to the hypothesis that GPI-anchored marker-deficient RBCs are true Pig-a mutants.