title: Effective use of the Pig-a gene mutation assay for mutagenicity screening: measuring CD59-deficient red blood cells in rats treated with genotoxic chemicals.
abstract: The Pig-a gene mutation assay using perpherial blood erythrocytes is being investigated as a screening tool for assessing mutagenicity in vivo. In this study, we evaluated two distinct approaches for performing the Pig-a assay in rats. We used antibodies to CD45 or the erythroid marker HIS49 to identify red blood cells (RBCs), and then monitored the kinetics of Pig-a mutant frequency, as measured by the frequency of CD59-deficient RBCs, in rats treated with the genotoxic chemicals, N-ethyl-N-nitrosourea, cyclophosphamide, 4-nitroquinoline-1-oxide, and ethylmethanesulfonate. In some instances, micronucleus frequency also was measured in the same animals. Time- and dose-related increases in Pig-a mutant frequency were found in all the chemical-treated groups, except for the groups treated with cyclophosphamide, which was a potent inducer of micronuclei. The two different approaches we employed were comparable for measuring induced mutant frequencies, but our historical data showed that the mean background frequencies for the CD45/CD59 method and the HIS49/CD59 method were 12.7 × 10(-6) and 5.5 ×10(-6), respectively. The relatively low, stable background mutant frequency associated with the HIS49/CD59 method indicates that it may have greater power for discriminating weak induced responses. These results suggest that the HIS49/CD59 method is a promising tool for measuring Pig-a mutant RBCs. In addition, differences in their manifestation kinetics and in their relative sensitivity for detecting different test compounds suggest that the combination of the Pig-a assay and the micronucleus assay may be effective in identifying in vivo genotoxicity.
authors.affiliation: TEIJIN Pharma Limited, 4-3-2., Asahigaoka, Hino Tokyo, Japan. t.kimoto@teijin.co.jp
authors.forename: Takafumi
authors.initials: T
authors.lastname: Kimoto
authors.forename: Satsuki
authors.initials: S
authors.lastname: Chikura
authors.forename: Kumiko
authors.initials: K
authors.lastname: Suzuki-Okada
authors.forename: Xiao-Mei
authors.initials: XM
authors.lastname: Kobayashi
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Itano
authors.forename: Daishiro
authors.initials: D
authors.lastname: Miura
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Kasahara
mesh_terms: D000818:Animals; D000906:Antibodies / Q000276:immunology*; D018957:CD59 Antigens / Q000276:immunology*; D004912:Erythrocytes / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D009152:Mutagenicity Tests / Q000379:methods*; D009153:Mutagens / Q000633:toxicity*; D051381:Rats
chemical_list: D000906:Antibodies; D018957:CD59 Antigens; D008565:Membrane Proteins; D009153:Mutagens; C080086:phosphatidylinositol glycan-class A protein