CD59‐deficient bone marrow erythroid cells from rats treated with procarbazine and propyl‐nitrosourea have mutations in the Pig‐a gene

• Published in: Environmental and molecular mutagenesis Vol. 61; no. 8; pp. 797 - 806
• Main Authors: Revollo, Javier R., Dad, Azra, Pearce, Mason G., Mittelstaedt, Roberta A., Casildo, Andrea, Lapidus, Rena G., Robison, Timothy W., Dobrovolsky, Vasily N.
• Format: Journal Article
• Language: English
• Published: Hoboken, USA John Wiley & Sons, Inc 01.10.2020; Wiley Subscription Services, Inc
• Subjects: Amino acid sequence; Animals; Antineoplastic Agents - toxicity; Bone marrow; Bone Marrow Cells - drug effects; Bone Marrow Cells - immunology; Carcinogens; CD59 antigen; CD59 Antigens - genetics; Deficient mutant; Erythrocytes; Erythroid cells; Flow cytometry; Genotype & phenotype; glycosyl phosphatidylinositol; Male; Membrane Proteins - genetics; Mutagens; Mutation; Next-generation sequencing; Nitrosourea Compounds - toxicity; Osteoprogenitor cells; Peripheral blood; Phenotypes; Point mutation; Procarbazine; Procarbazine - toxicity; Rats; Rats, Inbred F344; Rats, Sprague-Dawley; red blood cells; sequencing; Swine; red blood cells; glycosyl phosphatidylinositol; flow cytometry; sequencing
• ISSN: 0893-6692; 1098-2280; 1098-2280
• DOI: 10.1002/em.22402

Procarbazine (PCZ) and N‐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 RBC Pig‐a assay. In the present study, we traced the origin of the RBC mutant phenotype by analyzing Pig‐a mutations 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‐linked Pig‐a gene by next generation sequencing. Pig‐a mutations 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 the Pig‐a gene, 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 the Pig‐a gene. The causal relationship between BMEs and RBCs and the agent‐specific mutational spectra in CD59‐deicient BMEs indicate that the rat RBC Pig‐a assay, scoring CD59‐deficient mutant‐phenotype RBCs in peripheral blood, detects Pig‐a gene mutation.