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.

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 >1×106 RETs 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.

Evaluation of the RBC Pig-a and PIGRET assays using single doses of hydroxyurea and melphalan in rats.

To evaluate the suitability of the rat Pig-a assay on reticulocytes (PIGRET assay) as a short-term test, red blood cell (RBC) Pig-a and PIGRET assays after single doses with hydroxyurea (HU) and melphalan (L-PAM) were conducted and the results of both assays were compared. HU was administered once orally to male SD rats at 250, 500 and 1000mg/kg, and both assays were conducted using peripheral blood withdrawn from the jugular vein at 1, 2 and 4 weeks after dosing. L-PAM was administered at 1.25, 2.5 and 5mg/kg in the same manner. L-PAM produced significant dose-dependent increases in mutant frequencies in the PIGRET assay after single oral doses, but did not produce dose-dependent increases in mutant frequencies in the RBC Pig-a assay. These results suggest that the PIGRET assay is more sensitive for the evaluation of the mutagenic potential of L-PAM than the RBC Pig-a assay. In contrast, HU, a clastogenic but not DNA-reactive compound, gave negative results in both assays. The results with these 2 chemicals indicate that the single-dose PIGRET assay in rats has the potential to properly detect DNA-reactive compounds that directly cause DNA damage in a short-term assay.

Evaluation of the PIGRET assay in rats by single oral dosing with azidothymidine.

In vivo phosphatidylinositol glycan, class A (Pig-a) gene mutation assay using peripheral blood is known to be a novel and useful tool to evaluate the mutagenicity of compounds. Recently, the rat PIGRET assay which is an improved method for measuring Pig-a mutant cells in reticulocytes with magnetic enrichment of CD71 positive cells has been developed. Several reports showed that the PIGRET assay could detect the increase of Pig-a mutant frequency earlier than the Pig-a assay in total red blood cells (RBC Pig-a assay). Therefore, as part of a collaborative study by the Mammalian Mutagenicity Study (MMS) Group of the Japanese Environmental Mutagen Society, the usefulness of the PIGRET assay in comparison to the RBC Pig-a assay has been assessed for 24 compounds with various mechanisms of action. In the present study, we performed the PIGRET assay and RBC Pig-a assay with a nucleoside analogue, azidothymidine (AZT), and compared the results in these assays. We administered a single dose of AZT to rats by oral gavage up to 2000mg/kg and examined Pig-a mutant frequencies at days 7, 14 and 28 by PIGRET and RBC Pig-a assays. No significant increases in mutant frequency were observed after administration of AZT in both the RBC Pig-a and PIGRET assays and comparable to the previous results of the International Workshop on Genotoxicity Testing (IWGT) workgroup. AZT has been thought to induce not only DNA chain termination as a pharmacological effect but also a large deletion on the genome DNA. The Pig-a assays may be less sensitive to compounds such as AZT which induce large deletions on the genome DNA.

Evaluation of the RBC Pig-a assay and the PIGRET assay using benzo[a]pyrene in rats.

The red blood cell (RBC) Pig-a assay has the potential to detect the in vivo mutagenicity of chemicals. Recently, use of the Pig-a assay with reticulocytes (the PIGRET assay) reportedly enabled the in vivo mutagenicity of chemicals to be detected earlier than using the RBC Pig-a assay. To evaluate whether the PIGRET assay is useful and effective as a short-term test, compared with the RBC Pig-a assay, we performed both assays using benzo[a]pyrene (BP), which is a well-known mutagen. BP was used to dose 8-week-old male rats orally at 0, 75.0, 150, and 300mg/kg administered as a single administration. Peripheral blood samples were then collected on days 0, 7, 14, and 28 after treatment and were used in both assays. In the treatment groups receiving 150mg/kg of BP or more, both the RBC Pig-a assay and the PIGRET assay detected the in vivo mutagenicity of BP. In the 300mg/kg treatment group, in which a significant increase in the mutant frequency (MF) was observed at all the sampling points using both the RBC Pig-a assay and the PIGRET assay, the reticulocyte (RET) Pig-a MF was higher than the RBC Pig-a MF on days 7 and 14 after treatment; nevertheless, the negative control RET Pig-a MF was comparable to the negative control RBC Pig-a MF. In addition, the RET Pig-a MF began to increase after day 7 and reached a maximum value on day 14 after treatment, whereas the RBC Pig-a MF increased continuously from day 7 until day 28 after treatment. These results indicate that the PIGRET assay has a higher sensitivity than the RBC Pig-a assay and that the PIGRET assay is useful for the earlier detection of the in vivo mutagenicity of chemicals, compared with the RBC Pig-a assay.

Evaluation for a mutagenicity of aristolochic acid by Pig-a and PIGRET assays in rats.

The Pig-a assay, which uses the endogenous phosphatidylinositol glycan, class A gene (Pig-a) as a reporter of mutation, has been developed as a method for evaluating in vivo mutagenicity. Pig-a gene mutation can be detected by identifying the presence of CD59, the glycosylphosphatidylinositol anchor protein, on the surface of erythrocytes (RBC Pig-a assay) and reticulocytes (PIGRET assay). The International Workshop on Genotoxicity Testing (IWGT) showed the usefulness of the RBC Pig-a assay through the evaluation of several compounds. Aristolochic acid (AA), one of the evaluated compounds in the IWGT workgroup, is a carcinogenic plant toxin that is a relatively strong gene mutagen both in vitro and in vivo, but a weak inducer of micronuclei in vivo. In the present study, we examined the mutagenicity of AA in the peripheral blood of rats treated orally with a single dose of AA using Pig-a assays. Furthermore, we evaluated the advantages of the PIGRET assay compared with the RBC Pig-a assay. The results showed that a statistically significant increase in mutant frequency of the Pig-a gene was detected at day 28 by the RBC Pig-a assay, and at days 7, 14 and 28 by the PIGRET assay. In addition, the mutant frequency by the PIGRET assay was higher than that by the RBC Pig-a assay. These results indicate that the mutagenicity of AA can be detected using the Pig-a assays, as reported by the IWGT, and the PIGRET assay can detect Pig-a mutants at an early time point compared with the RBC Pig-a assay.

Evaluation of red blood cell Pig-a assay and PIGRET assay in rats using chlorambucil.

The Pig-a assay is a novel method to assess the in vivo mutagenicity of compounds, and it is expected to be useful for the detection of genotoxicity. In this study, to assess the performance of the Pig-a assay targeting red blood cells (RBCs; RBC Pig-a assay) and reticulocytes (RETs; PIGRET assay), chlorambucil, which is a genotoxicant, was orally administered to male rats once at 10, 20 and 40mg/kg on Day 1, and the mutant frequencies (MFs) of RBCs and RETs were examined periodically. In the RBC Pig-a assay, significant increases in MFs were observed at 40mg/kg on Day 15 and at 20mg/kg or higher on Day 29. In the PIGRET assay, MFs increased significantly at all dose levels on Day 8 and only at 20mg/kg on Day 15, but there was no increase in MFs in the treatment groups on Day 29. In conclusion, the RBC Pig-a assay and PIGRET assay in rats have sufficient sensitivity to detect the mutagenicity of chlorambucil, and the PIGRET assay could detect its mutagenicity earlier and at a lower dose than the RBC Pig-a assay.

Evaluation of single-dose RBC Pig-a and PIGRET assays in detecting the mutagenicity of thiotepa in rats.

The Pig-a assay, which uses reticulocytes (PIGRET assay) as target cells, is anticipated to detect mutagenicity at earlier time points than the RBC Pig-a assay, which uses all red blood cells as target cells. As part of a collaborative study conducted by the Mammalian Mutagenicity Study (MMS) Group, we evaluated the PIGRET and RBC Pig-a assays to detect Pig-a gene mutations induced by the carcinogen thiotepa. A single dose of thiotepa at 7.5, 15, and 30mg/kg was administered to 8-week-old male Sprague-Dawley rats by oral gavage. PIGRET and RBC Pig-a assays were performed using peripheral blood collected from rats 7, 14, and 28days after thiotepa administration (Day 0 as the day of administration), and the resulting Pig-a mutant frequencies (MFs) were compared. Increased Pig-a MF was observed from Day 7 onwards using the PIGRET assay. Pig-a MF remained fairly constant thereafter until Day 28 in the 30mg/kg group, whereas it peaked on Day 14 in the 7.5 and 15mg/kg groups. Using the RBC Pig-a assay, on the other hand, no significant increase in MF was observed at any of the dosages on Days 7, 14, or 28. These findings show that Pig-a gene mutations following a single dose of thiotepa were detected using the PIGRET assay but not the RBC Pig-a assay, which suggests that PIGRET assay is more suitable than RBC Pig-a assay for evaluating the in vivo mutagenicity by a single dose.

Evaluation of the PIGRET assay as a short-term test using a single dose of diethylnitrosamine.

The PIGRET assay, which was developed as the Pig-a assay in reticulocytes, can detect mutagenicity of compounds earlier than the Pig-a assay in total red blood cells (RBC; RBC Pig-a assay). The usefulness of the PIGRET assay as a short-term test has been confirmed in a collaborative study in Japan with 24 chemicals. One of these chemicals, diethylnitrosamine (DEN), which mainly induces liver tumors in both sexes of rats, was tested. To determine the appropriate doses, DEN was dissolved in physiological saline and administered orally with a single dose to male 8-week-old Sprague-Dawley rats in a preliminary dose-range finding study. As a result, all of the animals died at doses of 300 and 600mg/kg. Therefore, 37.5, 75, and 150mg/kg doses were set for the main study (the RBC Pig-a and PIGRET assays). The results showed no statistically significant increase in the mutant frequency (MF) of CD59-negative cells in the groups treated with DEN for the entire test period; however, the positive control N-ethyl-N-nitrosourea (ENU) produced positive results. Some hematogenic effects were indicated by the significant increase of the percentage of reticulocytes in the medium and at high doses on Day 28. The decrease in the body weight on Days 2-4 in the main study and the mortality in the preliminary dose range-finding study indicated that appropriate doses were used in the main study. Although DEN is a known genotoxic carcinogen in the liver, our negative results in the RBC Pig-a and PIGRET assays indicated that there is no substantial mutagenicity in hematopoietic cells under the conditions using a single dose. The PIGRET assay detected the mutagenicity of ENU one week earlier than the RBC Pig-a assay, indicating the usefulness of the PIGRET assay as a short-term test.

Results of rat Pig-a/PIGRET assay with a single dose regimen of 1,3-propane sultone and 2-acetyl aminofluorene.

The Pig-a assay is a useful in vivo mutation detecting test and is easier to perform than the in vivo transgenic mutation assay. This assay is now recognized to be able to detect a number of mutagenic chemicals administered to rats in sub-acute or sub-chronic dose studies. The present investigation was conducted to evaluate the usefulness of peripheral blood Pig-a assays with total red blood cells (RBC Pig-a assay) and with reticulocytes (PIGRET assay) using two genotoxic rodent carcinogens, 1,3-propane sultone (1,3-PS) and 2-acetylaminofluorene (2-AAF). Male rats were orally administered a single dose of each test compound, and both the RBC Pig-a and PIGRET assays were performed using flow cytometry to measure the Pig-a mutant frequency (MF) before and after dosing on Days 8, 15 and 29. In the experiment with 1,3-PS, significant increases in Pig-a MF were observed from Day 15 and Day 8 in the RBC Pig-a and PIGRET assays, respectively. The results of both assays demonstrated that the increases in Pig-a MF were detectable after a single treatment with 1,3-PS. Furthermore, the difference in the kinetics of the increase in Pig-a MF between the RBC Pig-a and PIGRET assays with 1,3-PS suggests that the PIGRET assay has an advantage in detecting the mutant erythrocytes earlier than the RBC Pig-a assay. In contrast, no significant increases were observed in the Pig-a assays using either RBC or reticulocytes with 2-AAF. The negative results in both assays with 2-AAF may indicate the limitation of the single dose method; however, further investigation at higher doses is necessary to determine limitation of the single dose method.