title: Analysis of mutations in the Pig-a gene of spleen T-cells from N-ethyl-N-nitrosourea-treated fisher 344 rats.
abstract: A rapid in vivo somatic cell gene mutation assay is being developed that measures mutation in the endogenous X-linked phosphatidylinositol glycan, class A gene (Pig-a). The assay detects Pig-a mutants by flow cytometric identification of cells deficient in glycosylphosphatidyl inositol (GPI) anchor synthesis. GPI-deficient, presumed Pig-a mutant cells also can be detected in a cloning assay that uses proaerolysin (ProAER) selection. Previously, we demonstrated that ProAER-resistant (ProAER(r) ) rat spleen T-cells have mutations in the Pig-a gene. In the present study, we report on a more complete analysis of ProAER(r) rat spleen T-cell mutants and describe a mutation spectrum for mutants isolated from rats 4 weeks after treatment with three consecutive doses of 35.6 mg/kg N-ethyl-N-nitrosourea (ENU). We identified a total of 55 independent mutations, with the largest percentage (69%) involving basepair substitution at A:T. The overall spectrum of Pig-a gene mutations was consistent with the types of DNA adducts formed by ENU and was very similar to what has been described for in vivo ENU-induced mutation spectra in other rodent reporter genes (e.g., in the endogenous Hprt gene and transgenic shuttle vectors). These data are consistent with the rat Pig-a assay detecting test-agent-induced mutational responses.
authors.affiliation: Teijin Pharma, Ltd, Tokyo, Japan.
authors.forename: Daishiro
authors.initials: D
authors.lastname: Miura
authors.forename: Joseph G
authors.initials: JG
authors.lastname: Shaddock
authors.forename: Roberta A
authors.initials: RA
authors.lastname: Mittelstaedt
authors.forename: Vasily N
authors.initials: VN
authors.lastname: Dobrovolsky
authors.forename: Takafumi
authors.initials: T
authors.lastname: Kimoto
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Kasahara
authors.forename: Robert H
authors.initials: RH
authors.lastname: Heflich
mesh_terms: D000818:Animals; D001427:Bacterial Toxins; D005038:Ethylnitrosourea / Q000633:toxicity*; D005434:Flow Cytometry; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D009152:Mutagenicity Tests / Q000379:methods*; D009154:Mutation; D052899:Pore Forming Cytotoxic Proteins; D051381:Rats; D011916:Rats, Inbred F344; D013154:Spleen / Q000166:cytology / Q000187:drug effects*; D013601:T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism
chemical_list: D001427:Bacterial Toxins; D008565:Membrane Proteins; D052899:Pore Forming Cytotoxic Proteins; C080086:phosphatidylinositol glycan-class A protein; C107096:proaerolysin; D005038:Ethylnitrosourea