Analysis of mutation in the rat Pig‐a assay: I) studies with bone marrow erythroid cells

Abstract

We have established a flow cytometry-based Pig-a assay for rat bone marrow erythroid cells (BMEs). The BME Pig-a assay uses a DNA-specific stain and two antibodies: one against the transmembrane transferrin receptor (CD71 marker) and the other against the GPI-anchored complement inhibitory protein (CD59 marker). In F344 male rats treated acutely with a total of 120 mg/kg of N-ethyl-N-nitrosourea (ENU) the frequency of CD59-deficient phenotypically mutant BMEs increased approximately 24-fold compared to the rats concurrently treated with the vehicle. Such an increase of mutant BMEs coincides with increases of CD59-deficient reticulocytes measured in rats treated with similar doses of ENU. Sequence analysis of the endogenous X-linked Pig-a gene of CD59-deficient BMEs revealed that they are Pig-a mutants. The spectrum of ENU-induced Pig-a mutations in these BMEs was consistent with the in vivo mutagenic signature of ENU: 73% of mutations occurred at A:T basepairs, with the mutated T on the nontranscribed strand of the gene. T→A transversion was the most frequent mutation followed by T→C transition; no deletion or insertion mutations were present in the spectrum. Since BMEs are precursors of peripheral red blood cells, our findings suggest that CD59-deficient erythrocytes measured in the flow cytometric erythrocyte Pig-a assay develop from BMEs containing mutations in the Pig-a gene. Thus, the erythrocyte Pig-a assay detects mutation in the Pig-a gene. Environ. Mol. Mutagen. 59:722-732, 2018. © 2018 Wiley Periodicals, Inc.

Analysis of mutation in the rat Pig‐a assay: II. Studies with bone marrow granulocytes

“…Ten or 12 days after the final treatment, the animals were sacrificed in pairs (control and ENU‐treated), and their tibias and femurs were harvested for collecting bone marrow cells. Most of the animals in this study were the same as in the study described by Revollo et al ().…”

“…Bone marrow was released from the tibias and femurs with 4 mL of 100% FBS supplemented with 2 mM Na 2 EDTA into a 5‐mL microcentrifuge tube as described in Revollo et al (). The flushed bone marrow was filtered through a 70‐μM nylon mesh sterile cell strainer (Fisherbrand, Pittsburgh, PA) into a 50‐mL centrifuge tube, diluted with 20 mL of ice‐cold PBS + 2% FBS (modified PBS) and divided into two halves.…”

“…The basic preparation used for cell staining with antibodies for analytical samples and for cell sorting or for flow cytometry analyses (e.g., centrifugations, filtering, cytometer setting, etc.) were as described in Revollo et al ().…”

“…1 × 10 5 CD11b + cells were sorted on a FACSAria sorter using the purity mode into modified PBS. The sorted cells were deposited onto a glass microscope slide and stained with 10% Wright Giemsa and 10% Toluidine Blue in a covered Coplin jar as in Revollo et al (). Cells were observed with a 100× objective on an upright Nikon 80i microscope and photographed using an attached digital Nikon DS‐Fi1 color camera and Elements software (Nikon, Melville, NY).…”

“…Lymphocytes and erythrocytes both originate from the same hemocytoblast stem cells in bone marrow, although they develop through different lineage progenitors (i.e., lymphoid and myeloid progenitors, respectively) (Schmaier, ). Furthermore, treatment with ENU also induces mutations in precursors of peripheral blood erythrocytes and bone marrow erythroid cells (BMEs), both in mice and rats (Kimoto et al, ; Revollo et al, ). Granulocytes are developmentally related to erythrocytes, both originating from the myeloid lineage of hematopoietic cells.…”