The phosphatidylinositol glycan anchor biosynthesis, Class A gene (Pig-a in rodents, PIG-A in humans) codes for a catalytic subunit of the N-acetylglucosamine transferase complex that is involved in an early step of glycosylphosphatidyl inositol (GPI) anchor synthesis, and GPI anchors tether specific protein markers to the surface of various types of cells. Two distinct strategies for Pig-a gene mutation assay used peripheral blood have been developed, one using flow cytometry and the other using limiting-dilution cloning. The limiting-dilution cloning assay using bacterial protoxin, proaerolysin, as a selective agent is resource intensive. For routine analysis of mutant frequency, the flow cytometric procedures employing fluorescently labeled antibodies against GPI-anchored markers (e.g., anti-CD59 for rat red blood cells, anti-CD24 for mouse red blood cells) are preferred for routine analysis of mutant frequency. The advantage of the cloning assay, however, is in-depth analyses of the mutant genotype (mutational spectra analysis). The characteristics of the flow cytometric Pig-a gene mutation assays, i.e., induced Pig-a mutant frequencies were persistence and the effect of split doses of chemicals were additive, make the assay on reticulocytes and total red blood cells an attractive possibility for developing detailed mutagenicity data in vivo. Although a large amount of information must be gathered on assay performance, progress toward the goal in the last few years has been rapid, and multi-laboratory trial has been initiated and ongoing.

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The in vivo mutagenesis from ethyl methanesulfonate (EMS) was investigated by the original Pig-a assay, which measures Pig-a mutant cells in red blood cells, and the PIGRET assay, which uses reticulocytes as a part of a collaborative study supported by the Japan Health Sciences Foundation. For the studies with the Pig-a assay, three dose levels of EMS (25, 50 and 100 mg/kg/day) were given orally to 6-week-old male F344 rats for 28 days, and peripheral blood was sampled just before the start of dosing, and after dosing for 1, 2 and 4 weeks with EMS. In the studies with the PIGRET assay, single oral dosing at two dose levels (360 and 720 mg/kg) of EMS was employed and peripheral blood was collected just before dosing, and 1 and 2 weeks after dosing of EMS. As the results, a statistically significant increase in mutant frequency of the Pig-a gene was observed at 2 and 4 weeks, but not at one week after dosing of EMS in the Pig-a assay. In the PIGRET assay, on the other hand, a statistically significant increase in mutant frequency was obtained at one week after the dosing. These results indicate that the PIGRET assay can detect Pig-a mutants much earlier than the original Pig-a assay can by focusing on reticulocytes rather than red blood cells as the target cell population.

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The Pig-a gene is involved in the synthesis of glycosylphosphatidylinositol (GPI) anchors. Pig-a gene mutations can be detected by identifying the presence of CD59, the GPI anchor protein, on the surface of erythrocytes (RBC Pig-a assay) and reticulocytes (PIGRET assay) and can be identified using flow cytometry. The usefulness of these Pig-a gene mutation assays has been confirmed in multi-laboratory trials with referenced mutagens. Although 4,4′-methylenedianiline (MDA) is an aromatic amine and has been identified as a potent hepatic carcinogen, in vivo micronucleus tests for MDA in hematopoietic cells determined that it was negative to weakly positive for genotoxicity. In the present study, we examined the mutagenicity of MDA in the peripheral blood of rats after 1- and 28-day MDA dosing using the Pig-a gene mutation assays. We also examined the utility of the RBC Pig-a and PIGRET assays. No changes in mutation frequency were observed after one-day MDA administration. Repeated dosing caused a moderate increase in mutation frequency compared to vehicle control at days 14 and 28, as measured by the RBC Pig-a assay and at day 14 by the PIGRET assay. The highest mutation frequency was found on days 7 and 14 by the PIGRET and RBC Pig-a assays, respectively.
In this study, we detected the mutagenicity of MDA in peripheral blood samples using gene mutation assays and judged to be positive for the MDA mutagenicity since a significant increase in mutation frequency was observed at high dose. These assays are expected to be easily integrated into general toxicity tests and to be combined with existing genotoxicity studies.

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A recent report on an interlaboratory trial on the Pig-a mutation assays using an antibody against the erythroid marker (HIS49 Pig-a assays) has shown transferability and reproducibility of the assays. In this report, methyl methanesulfonate (MMS) was used to further evaluate the ability of HIS49 Pig-a assays in different study designs (28-day repeated dose and single dose). MMS was administered to male Sprague Dawley (SD) rats orally for 28 days at a dose of 7.5, 15, or 30 mg/kg/day or by single oral gavage at 50, 100 and 200 mg/kg in 28-day study and single dose study, respectively. Pig-a assays for total red blood cells (RBC Pig-a assay), for reticulocytes (PIGRET assay) and micronucleated reticulocytes (MN-RET) measurement were performed. In the 28-day study, there were significant increases in the Pig-a mutant frequency (Pig-a MF) in PIGRET assay at 30 mg/kg/day on Days 8, 15 and 29 with the maximum Pig-a MF on Day 29. On the contrary, there was no change in the Pig-a MF in RBC Pig-a assay. In the single dose study, MMS resulted in a steady increase in Pig-a MF in PIGRET assay from Day 8 through to Day 29 and a delayed increase in Pig-a MF in RBC Pig-a assay at Day 15 and 29 with maximam Pig-a MF on Day 29. MMS also induced significant increases in the MN-RET frequency on Day 4 of the 28-day study and on Day 3 of the single dose study. These results suggest that the PIGRET assay has the following advantages. The accumulated properties with repeated doses would be useful when the assay is integrated with a repeated dose general toxicity study, and the rapid detection of mutagenesis using a single dose protocol would be useful for early assessment of the in vivo mutagenicity of chemicals.

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The peripheral blood Pig-a assay is now recognized as one of genetic toxicology test to detect the in vivo mutagenic potential of chemical. A previous report on interlaboratry trial by Japanese research group has shown that the rat Pig-a assay with an antibody binds to an erythroid marker is transferable and reproducible. By using this approach, we evaluated the capability of the Pig-a assay protocol to integrate into the general toxicity studies (single or repeated dose study). Both Pig-a assay in total red blood cells (RBC Pig-a assay) and Pig-a assay in reticulocytes (PIGRET assay) were performed before and at days 8, 15 and 29 following single or 28-daily treatments of cyclophosphamide (CP). The difference in the kinetics of increase in Pig-a mutant frequency (MF) between total red blood cell (RBC) and reticulocyte (RET) was found in the single dose study; RET Pig-a MF was temporary increased at days 8 and 15, while RBC Pig-a MF was increased only at day 15. In the repeated dose study, the RET Pig-a MF was increased in the high dose group at day 29, though it was the result under the conditional statistical analysis which excluded one outlier in the control group. The manuscript by Dertinger et al, also showed the increase of Pig-a MFs in both RBCs and RETs, suggesting that the Pig-a assay for the repeated dose study is feasible to detect the mutagenicity of CP. Taken together, the increase of Pig-a MF was detectable under the both single and 28-day repeated dose study with CP. These results suggest that the Pig-a assay approaches are practical in the general toxicity studies. In addition, the PIGRET assay is an advantageous method at the point that the increase in mutant cells is more detectable at an early stage compared with the RBC Pig-a assay. It is thought that this phenomenon is based on the differentiation stage of an erythroid lineage.

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The emerging Pig-a gene mutation assay, a powerful and promising tool for evaluating in vivo genotoxicity, is based on flow cytometric enumeration of red blood cells (RBCs), which are deficient in glycosylphosphatidylinositol anchored protein. Various approaches for measuring Pig-a mutant cells have been developed, particularly those focused on peripheral RBCs and reticulocytes (RETs). Previously, it had been reported that Pig-a and gpt mutant frequencies were relatively increased in N-ethyl-N-nitrosourea (ENU)- and benzo[a]pyrene (BP)-treated mice. The capacity and characteristics of the Pig-a assay relative to transgenic rodent (TGR) mutation assays, however, are unclear in rats. Here, using transgenic gpt delta rats, we compared the in vivo genotoxicity of single oral doses of ENU (40 mg/kg) in the gpt gene mutation assay in bone marrow and liver, and Pig-a gene mutation assays on RBCs and RETs in the same animals. The Pig-a gene mutation assays were conducted at 1, 2, and 4 weeks after treatment, whereas gpt assays were conducted on tissues collected at the 4-week terminal sacrifice. Consequently, we detected that Pig-a and gpt mutant frequencies were clearly increased in ENU-treated rats, indicating that both the Pig-a and TGR gene mutation assays can detect in vivo ENU genotoxicity equally.

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