title: CD48-deficient T-lymphocytes from DMBA-treated rats have de novo mutations in the endogenous Pig-a gene.
abstract: A major question concerning the scientific and regulatory acceptance of the rodent red blood cell-based Pig-a gene mutation assay is the extent to which mutants identified by their phenotype in the assay are caused by mutations in the Pig-a gene. In this study, we identified T-lymphocytes deficient for the glycosylphosphatidylinositol-anchored surface marker, CD48, in control and 7,12-dimethylbenz[a]anthracene (DMBA)-treated rats using a flow cytometric assay and determined the spectra of mutations in the endogenous Pig-a gene in these cells. CD48-deficient T-cells were seeded by sorting at one cell per well into 96-well plates, expanded into clones, and exons of their genomic Pig-a were sequenced. The majority (78%) of CD48-deficient T-cell clones from DMBA-treated rats had mutations in the Pig-a gene. The spectrum of DMBA-induced Pig-a mutations was dominated by mutations at A:T, with the mutated A being on the nontranscribed strand and A → T transversion being the most frequent change. The spectrum of Pig-a mutations in DMBA-treated rats was different from the spectrum of Pig-a mutations in N-ethyl-N-nitrosourea (ENU)-treated rats, but similar to the spectrum of DMBA mutations for another endogenous X-linked gene, Hprt. Only 15% of CD48-deficient mutants from control animals contained Pig-a mutations; T-cell biology may be responsible for a relatively large fraction of false Pig-a mutant lymphocytes in control animals. Among the verified mutants from control rats, the most common were frameshifts and deletions. The differences in the spectra of spontaneous, DMBA-, and ENU-induced Pig-a mutations suggest that the flow cytometric Pig-a assay detects de novo mutation in the endogenous Pig-a gene.
authors.affiliation: Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, Arkansas.
authors.forename: Vasily N
authors.initials: VN
authors.lastname: Dobrovolsky
authors.affiliation: Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, Arkansas.
authors.forename: Javier
authors.initials: J
authors.lastname: Revollo
authors.affiliation: Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, Arkansas.
authors.forename: Mason G
authors.initials: MG
authors.lastname: Pearce
authors.affiliation: Departamento De Ciencias De La Salud, Universidad Autónoma Metropolitana Iztapalapa, Mexico City, Mexico.
authors.forename: M Monserrat
authors.initials: MM
authors.lastname: Pacheco-Martinez
authors.affiliation: Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, Arkansas.
authors.forename: Haixia
authors.initials: H
authors.lastname: Lin
mesh_terms: D015127:9,10-Dimethyl-1,2-benzanthracene / Q000633:toxicity*; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D000071178:CD48 Antigen; D005434:Flow Cytometry; D008297:Male; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D009152:Mutagenicity Tests; D009153:Mutagens / Q000633:toxicity; D009154:Mutation; D011916:Rats, Inbred F344; D013601:T-Lymphocytes / Q000187:drug effects* / Q000378:metabolism
chemical_list: D015703:Antigens, CD; D000071178:CD48 Antigen; C000604067:Cd48 protein, rat; D008565:Membrane Proteins; D009153:Mutagens; C080086:phosphatidylinositol glycan-class A protein; D015127:9,10-Dimethyl-1,2-benzanthracene
keywords: cell sorting; flow cytometry; glycosylphosphatidylinositol (GPI) anchor; sequencing