Effective use of the Pig-a gene mutation assay for mutagenicity screening: measuring CD59-deficient red blood cells in rats treated with genotoxic chemicals

• Published in: Journal of toxicological sciences Vol. 37; no. 5; pp. 943 - 955
• Main Authors: Kimoto, Takafumi, Chikura, Satsuki, Suzuki-Okada, Kumiko, Kobayashi, Xiao-mei, Itano, Yasuhiro, Miura, Daishiro, Kasahara, Yoshinori
• Format: Journal Article
• Language: English
• Published: Japan The Japanese Society of Toxicology 2012
• Subjects: Animals; Antibodies - immunology; CD59 Antigens - immunology; Erythrocytes - drug effects; Erythrocytes - metabolism; Flow cytometry; In vivo gene mutation assay; In vivo micronucleus assay; Indexing in process; Male; Membrane Proteins - genetics; Mutagenicity Tests - methods; Mutagens - toxicity; Peripheral red blood cells; Phosphatidylinositol glycan complementation class A gene; Rats
• ISSN: 0388-1350; 1880-3989
• DOI: 10.2131/jts.37.943

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.