Cd59-Deficient Bone Marrow Erythroid Cells From Rats Treated With Procarbazine And Propyl-Nitrosourea Have Mutations In Thepig-Agene

Procarbazine (PCZ) andN-propyl-N-nitrosourea (PNU) are rodent mutagens and carcinogens. Both induce GPI-anchored marker-deficient mutant-phenotype red blood cells (RBCs) in the flow cytometry-based rat RBCPig-aassay. In the present study, we traced the origin of the RBC mutant phenotype by analyzingPig-amutations in the precursors of RBCs, bone marrow erythroid cells (BMEs). Rats were exposed to a total of 450 mg/kg PCZ hydrochloride or 300 mg/kg PNU, and bone marrow was collected 2, 7, and 10 weeks later. Using a flow cell sorter, we isolated CD59-deficient mutant-phenotype BMEs from PCZ- and PNU-treated rats and examined their endogenous X-linkedPig-agene by next generation sequencing.Pig-amutations consistent with the properties of PCZ and PNU were found in sorted mutant-phenotype BMEs. PCZ induced mainly A > T transversions with the mutated A on the nontranscribed strand of thePig-agene, while PNU induced mainly T > A transversions with the mutated T on the nontranscribed strand. The treatment-induced mutations were distributed across the protein coding sequence of thePig-agene. The causal relationship between BMEs and RBCs and the agent-specific mutational spectra in CD59-deicient BMEs indicate that the rat RBCPig-aassay, scoring CD59-deficient mutant-phenotype RBCs in peripheral blood, detectsPig-agene mutation.