title: The PIGRET assay, a method for measuring Pig-a gene mutation in reticulocytes, is reliable as a short-term in vivo genotoxicity test: Summary of the MMS/JEMS-collaborative study across 16 laboratories using 24 chemicals.
abstract: The in vivo mutation assay using the X-linked phosphatidylinositol glycan class A gene (Pig-a in rodents, PIG-A in humans) is a promising tool for evaluating the mutagenicity of chemicals. Approaches for measuring Pig-a mutant cells have focused on peripheral red blood cells (RBCs) and reticulocytes (RETs) from rodents. The recently developed PIGRET assay is capable of screening >1×106RETs for Pig-a mutants by concentrating RETs in whole blood prior to flow cytometric analysis. Additionally, due to the characteristics of erythropoiesis, the PIGRET assay can potentially detect increases in Pig-a mutant frequency (MF) sooner after exposure compared with a Pig-a assay targeting total RBCs (RBC Pig-a assay). In order to test the merits and limitations of the PIGRET assay as a short-term genotoxicity test, an interlaboratory trial involving 16 laboratories was organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagenicity Society (MMS/JEMS). First, the technical proficiency of the laboratories and transferability of the assay were confirmed by performing both the PIGRET and RBC Pig-a assays on rats treated with single doses of N-nitroso-N-ethylurea. Next, the collaborating laboratories used the PIGRET and RBC Pig-a assays to assess the mutagenicity of a total of 24 chemicals in rats, using a single treatment design and mutant analysis at 1, 2, and 4 weeks after the treatment. Thirteen chemicals produced positive responses in the PIGRET assay; three of these chemicals were not detected in the RBC Pig-a assay. Twelve chemicals induced an increase in RET Pig-a MF beginning 1 week after dosing, while only 3 chemicals positive for RBC Pig-a MF produced positive responses 1 week after dosing. Based on these results, we conclude that the PIGRET assay is useful as a short-term test for in vivo mutation using a single-dose protocol.
authors.affiliation: Teijin Pharma Limited, 4-3-2 Asahigaoka, Hino-shi, Tokyo, 191-8512, Japan. Electronic address: t.kimoto@teijin.co.jp.
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authors.forename: Yohei
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authors.affiliation: National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo, 158-0098, Japan.
authors.forename: Takeshi
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authors.affiliation: National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo, 158-0098, Japan.
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mesh_terms: D004912:Erythrocytes / Q000187:drug effects; D005038:Ethylnitrosourea / Q000633:toxicity; D006801:Humans; D007373:Interinstitutional Relations; D007753:Laboratories / Q000458:organization & administration*; D008565:Membrane Proteins / Q000235:genetics*; D009152:Mutagenicity Tests / Q000379:methods*; D009154:Mutation; D015203:Reproducibility of Results; D012156:Reticulocytes / Q000187:drug effects*
chemical_list: D008565:Membrane Proteins; C080086:phosphatidylinositol glycan-class A protein; D005038:Ethylnitrosourea
keywords: Gene mutation; PIGRET; Pig-a assay; Reticulocyte