Analysis of mutations in the Pig-a gene of spleen T-cells from N-ethyl-N-nitrosourea-treated fisher 344 rats

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...

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Bibliographic Details
Published in:Environmental and molecular mutagenesis 2011-06, Vol.52 (5), p.419-423
Main Authors: Miura, Daishiro, Shaddock, Joseph G., Mittelstaedt, Roberta A., Dobrovolsky, Vasily N., Kimoto, Takafumi, Kasahara, Yoshinori, Heflich, Robert H.
Format: Article
Language:English
Subjects:
Animals
Bacterial Toxins
Biological and medical sciences
class A gene
Ethylnitrosourea - toxicity
Flow Cytometry
Fundamental and applied biological sciences. Psychology
Genetics of eukaryotes. Biological and molecular evolution
in vivo gene mutation assay
Male
Medical sciences
Membrane Proteins - genetics
Mutagenicity Tests - methods
Mutation
mutation spectrum
N-ethyl-N-nitrosourea
phosphatidylinosital glycan
Pore Forming Cytotoxic Proteins
proaerolysin
Rats
Rats, Inbred F344
Spleen - cytology
Spleen - drug effects
T-Lymphocytes - drug effects
T-Lymphocytes - metabolism
Toxicology
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Summary: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 (ProAERr) rat spleen T‐cells have mutations in the Pig‐a gene. In the present study, we report on a more complete analysis of ProAERr 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. Environ. Mol. Mutagen., 2011. Published 2011 Wiley‐Liss, Inc.
ISSN:0893-6692
1098-2280
DOI:10.1002/em.20654