title: Development of an in vivo gene mutation assay using the endogenous Pig-A gene: I. Flow cytometric detection of CD59-negative peripheral red blood cells and CD48-negative spleen T-cells from the rat.
abstract: The product of the phosphatidylinositol glycan complementation group A gene (Pig-A) is involved in the synthesis of glycosylphosphatidylinositol (GPI) anchors that link various protein markers to the surface of several types of mammalian cells, including hematopoietic cells. Previous observations indicate that Pig-A mutation results in the lack of GPI synthesis and the absence of GPI-anchored proteins on the cell surface. As a first step in designing a rapid assay for measuring Pig-A mutation in the rat, we developed flow cytometry (FCM) strategies for detecting GPI-negative cells in rat peripheral blood and spleen. Anti-CD59 was used to detect GPI-anchored proteins on red blood cells (RBCs), and anti-CD48 was used to detect GPI-anchored proteins on spleen T-cells. The spontaneous frequency of CD59-negative RBCs in five male F344 rats ranged from 1 x 10(-6) to 27 x 10(-6). In contrast, treatment of five rats with three doses of 40 mg/kg N-ethyl-N-nitrosourea (ENU) increased the frequency of CD59-negative RBCs to 183 x 10(-6) to 249 x 10(-6) at 2 weeks and to 329 x 10(-6) to 413 x 10(-6) at 4 weeks after dosing. In the same 4-week posttreatment rats, the frequency of CD48-negative T-cells was 11 x 10(-6) to 16 x 10(-6) in control rats and 194 x 10(-6) to 473 x 10(-6) in ENU-treated rats. The frequencies of GPI-deficient cells were similar for RBCs and spleen T-cells. These results indicate that FCM detection of GPI-linked markers may form the basis for a rapid in vivo mutation assay. Although RBCs may be useful for a minimally invasive assay, T-cells are a promising tissue for both detecting GPI-deficient cells and confirming that Pig-A gene mutation is the cause of the phenotype.
authors.affiliation: Division of Genetic and Reproductive Toxicology, US Food and Drug Administration, National Center for Toxicological Research, Jefferson, Arkansas 72079, USA.
authors.forename: Daishiro
authors.initials: D
authors.lastname: Miura
authors.forename: Vasily N
authors.initials: VN
authors.lastname: Dobrovolsky
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Kasahara
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Katsuura
authors.forename: Robert H
authors.initials: RH
authors.lastname: Heflich
mesh_terms: D000818:Animals; D015703:Antigens, CD / Q000276:immunology*; D000071178:CD48 Antigen; D018957:CD59 Antigens / Q000097:blood*; D004912:Erythrocytes / Q000276:immunology*; D005038:Ethylnitrosourea / Q000633:toxicity; D005434:Flow Cytometry / Q000379:methods*; D017261:Glycosylphosphatidylinositols; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D009152:Mutagenicity Tests; D009153:Mutagens / Q000633:toxicity; D051381:Rats; D011916:Rats, Inbred F344; D013154:Spleen / Q000166:cytology*; D013601:T-Lymphocytes / Q000276:immunology*
chemical_list: D015703:Antigens, CD; D000071178:CD48 Antigen; D018957:CD59 Antigens; C000604067:Cd48 protein, rat; D017261:Glycosylphosphatidylinositols; D008565:Membrane Proteins; D009153:Mutagens; C080086:phosphatidylinositol glycan-class A protein; D005038:Ethylnitrosourea