Red Blood Cell Pig-a Assay Research Articles

CD59-deficient bone marrow erythroid cells from rats treated with procarbazine and propyl-nitrosourea have mutations in the Pig-a gene.

Procarbazine (PCZ) and N-propyl-N-nitrosourea (PNU) are rodent mutagens and carcinogens. Both induce GPI-anchored marker-deficient mutant-phenotype red blood cells (RBCs) in the flow cytometry-based rat RBC Pig-a assay. In the present study, we traced the origin of the RBC mutant phenotype by analyzing Pig-a mutations in the precursors of RBCs, bone marrow erythroid cells (BMEs). Rats were exposed to a total of 450 mg/kg PCZ hydrochloride or 300 mg/kg PNU, and bone marrow was collected 2, 7, and 10 weeks later. Using a flow cell sorter, we isolated CD59-deficient mutant-phenotype BMEs from PCZ- and PNU-treated rats and examined their endogenous X-linked Pig-a gene by next generation sequencing. Pig-a mutations consistent with the properties of PCZ and PNU were found in sorted mutant-phenotype BMEs. PCZ induced mainly A > T transversions with the mutated A on the nontranscribed strand of the Pig-a gene, while PNU induced mainly T > A transversions with the mutated T on the nontranscribed strand. The treatment-induced mutations were distributed across the protein coding sequence of the Pig-a gene. The causal relationship between BMEs and RBCs and the agent-specific mutational spectra in CD59-deicient BMEs indicate that the rat RBC Pig-a assay, scoring CD59-deficient mutant-phenotype RBCs in peripheral blood, detects Pig-a gene mutation.

Standard protocol for the total red blood cell Pig-a assay used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society

The Pig-a assay, a promising tool for evaluating in vivo genotoxicity, is based on flow cytometric enumeration of red blood cells (RBCs) that are deficient in glycosylphosphatidylinositol anchor protein. Various approaches for measuring Pig-a mutant cells have been developed, particularly focusing on measuring mutants in peripheral RBCs and reticulocytes (RETs). The Pig-a assay on concentrated RETs—the PIGRET assay—has the potential to detect genotoxicity in the early stages of a study. To verify the potential and usefulness of the PIGRET assay for short-term testing, we conducted an interlaboratory trial involving 16 laboratories organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society (MMS/JEMS). The collaborating laboratories assessed the mutagenicity of a total of 24 chemicals in rats using a single-treatment design and standard protocols for conducting the Pig-a assay on total RBCs (the RBC Pig-a assay) and the PIGRET assay. Here, we describe the standard protocol for the RBC Pig-a assay in detail.

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 a single-dose PIGRET assay for cisplatin in rats compared with the RBC Pig-a assay

As a part of a collaborative study of the Pig-a assay by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society, a genotoxicity study on cisplatin was performed using red blood cell (RBC) Pig-a and PIGRET assays. The dose levels were set at 0 (vehicle, physiological saline), 0.5, 1, and 2 mg/kg, and cisplatin was administered intravenously once to male F344 rats. The RBC Pig-a and PIGRET assays were performed using peripheral blood collected at pre-dosing and 1, 2 and 4 weeks after dosing. In the RBC Pig-a assay, an increase in the Pig-a mutant frequency (MF) was observed at week 4 in the high dose group. Although a significant increase in the Pig-a MF was also observed at week 2 in all cisplatin-treated groups, it was considered that this change was caused by a low MF in the vehicle control group and not to be biologically relevant. In the PIGRET assay, the Pig-a MF was increased at weeks 1, 2 and 4 in the high dose group. In addition, the means of the vehicle control group’s Pig-a MFs in the PIGRET assay were lower than those in the RBC Pig-a assay. Based on the above results, cisplatin was determined to have mutagenicity under the conditions of this study, and it was demonstrated that the PIGRET assay was an appropriate tool to evaluate the in vivo mutagenicity much earlier than the RBC Pig-a assay.

Evaluation of the in vivo mutagenicity of melamine by the RBC Pig-a assay and PIGRET assay

The Pig-a assay is a new in vivo genotoxicity test for detecting mutagens in the bodies of animals, using the endogenous Pig-a gene as the target. There are two types of Pig-a assays: the red blood cell (RBC) Pig-a assay, which uses RBCs, and the PIGRET assay, which uses reticulocytes. The Japanese Environmental Mutagen Society-Mammalian Mutagenicity Study Group collaborative study of the Pig-a assay was carried out to investigate the usefulness of the PIGRET assay. The mutagenicity of melamine was evaluated as part of this study. Eight-week-old male Crl:CD (SD) rats were administered a single gavage dose of melamine as a non-genotoxic bladder carcinogen. Blood samples were collected at the first, second and fourth weeks after administration, and the RBC Pig-a assay and PIGRET assays were conducted using these samples. Three dose levels were used in the study: the highest dose was 2000mg/kg, which is generally used as the maximum dose in in vivo genotoxicity testing, and 1000 and 500mg/kg were also used. As a positive control, a group of rats was administered a single dose of N-nitroso-N-ethylurea (ENU) by gavage at 40mg/kg. The Pig-a mutant frequencies (Pig-a MFs) did not increase in any of the melamine groups throughout the experimental period in either the RBC Pig-a assay or the PIGRET assay. Both the RBC Pig-a and PIGRET assays revealed significant increases in the Pig-a MFs in the ENU group, starting at day 7 after a single administration. Therefore, these two assays, when evaluated after a single administration, can be used to determine that melamine is non-mutagenic.

Pyrene did not induce gene mutation in red blood cell Pig-a assay and PIGRET assay in rats

A new in vivo gene mutation assay has been developed based on the phosphatidylinositol glycan anchor biosynthesis, Class A gene (Pig-a in rodents) as an endogenous reporter. Although a large number of chemicals have been evaluated in the rat Pig-a assay in 28-day repeat dose regimens, there was limited reporting of rat Pig-a assay after a single dose. A collaborative study by the Mammalian Mutagenicity Study group, which is a subgroup of the Japanese Environmental Mutagen Society, was conducted to verify the usefulness of the rat Pig-a assay after a single dose as a short-term genotoxicity test. As a part of this collaborative study, the in vivo mutagenicity of a single dose of pyrene (Pyr) was investigated in the red blood cell (RBC Pig-a assay) and in reticulocytes (PIGRET) of rats. Eight-week old male rats were orally dosed with Pyr at 500, 1000, and 2000 mg/kg or ethylnitrosourea (ENU) at 10 and 40 mg/kg as a positive control. The animals in each group were examined for Pig-a mutant frequencies (MF) except for animals in the 2000mg/kg group because of mortality or severe toxicity. The Pig-a MF in RBCs and reticulocytes, as CD59 negative cells, were evaluated once a week for 4 weeks after the dosing. With a single exposure to ENU, the Pig-a MF in both RBCs and reticulocytes increased in a time- and dose-dependent manner. In contrast, no statistically significant effect was observed in rats dosed with Pyr at 500 and 1000 mg/kg. Therefore, Pyr was concluded to be negative in the RBC Pig-a assay and the PIGRET assay after a single oral administration in rats. The result was consistent with previously reported Pig-a assays with repeat dose regimens.

The rat Pig-a assay using an erythroid HIS49 antibody in a single dose study of isopropyl p-toluenesulfonate

As part of a collaborative study in the Mammalian Mutagenicity Study group of the Japanese Environmental Mutagen Society, we evaluated the in vivo mutagenicity of isopropyl p-toluenesulfonate (IPTS) using a peripheral blood Pig-a assay in rats. Pig-a mutant frequency (MF) data was obtained for both red blood cells (RBCs) and reticulocytes (RETs) at 1, 2 and 4 weeks after a single oral administration of IPTS at doses of 125, 250, or 500mg/kg. The results of the RBC Pig-a assay demonstrated that both the 250 and 500mg/kg treatment groups showed significant increases in Pig-a MF only at 4 weeks after IPTS treatment. In comparison, the PIGRET assay showed a clear and dose-related increase in Pig-a MF at 1 week after treatment, with a continuous increase until 4 weeks after treatment observed in the highest dose group. These results indicate that the both the RBC Pig-a assay and PIGRET assay can detect in vivo IPTS mutagenicity under a single dosing protocol. In particular, the PIGRET assay, which uses magnetic enrichment to analyze greater numbers of RETs in a high-throughput manner, showed an increase in Pig-a MF earlier than the RBC Pig-a assay. The PIGRET assay is also considered to be more sensitive than the RBC Pig-a assay because it exhibits a low spontaneous Pig-a MF. For this reason, the PIGRET assay clearly identified small increases in Pig-a MF as significant at the lower doses than in the RBC Pig-a assay under the conditions in this study.

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 of a single-dose PIGRET assay for acetaminophen in rats compared with the RBC Pig-a assay

As a part of a collaborative study of the Pig-a assay by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society, a genotoxicity study on acetaminophen (APAP) was performed using the red blood cell (RBC) Pig-a and PIGRET assays. The dose levels were set at 0 (vehicle, 0.5% methylcellulose solution), 500, 1000, and 2000mg/kg, and APAP was administered once by oral gavage to male Sprague Dawley rats. For the positive control group, N-nitroso-N-ethylurea (ENU, 40mg/kg) was administered in the same way. The RBC Pig-a and PIGRET assays were performed using peripheral blood collected at pre-dosing and 1, 2 and 4 weeks after dosing. In both the RBC Pig-a and PIGRET assays, there were no changes in the Pig-a gene mutant frequency (MF) by the APAP treatment at any time point. The Pig-a MFs as measured by the RBC Pig-a assay for the ENU-treated group increased in a time-dependent manner with the maximum value at week 4; however, those using the PIGRET assay reached comparable values at week 1. Based on the above results, APAP was determined to have no mutagenicity under the conditions of this study, and the PIGRET assay could detect mutagenicity of ENU much earlier than the RBC Pig-a assay.

Red blood cell Pig-a assay and PIGRET assay in rats with azathioprine

A new in vivo gene mutation assay has been developed based on the phosphatidylinositol glycan anchor biosynthesis, Class A gene (Pig-a in rodents) as an endogenous reporter. Using this Pig-a assay, the in vivo mutagenicity of a single dose of azathioprine (Aza) was investigated in red blood cells (RBC Pig-a assay) and reticulocytes (PIGRET) of rats. Eight-week old male rats were orally dosed once with Aza at 50, 100 and 200mg/kg or ethylnitrosourea (ENU) at 10 and 40mg/kg as a positive control. Because 4 out of 6 animals at 200mg/kg of Aza died 3days after the dosing, this dose group was excluded for analyses. The frequencies of Pig-a mutants in RBCs and reticulocytes (RET) were evaluated once a week for 4 weeks after the treatment. With a single exposure to ENU, the frequencies of Pig-a mutants in both RBCs and RETs increased in a time- and dose-dependent manner. In contrast, with Aza small effects that were not statistically significant were observed in rats at 21 and 14days in the RBC Pig-a and PIGRET assays respectively. Based on the present results, the mutagenic potential of Aza is negligible after single oral administration in rats.

Evaluation of mutagenicity of acrylamide using RBC Pig-a and PIGRET assays by single peroral dose in rats

The Pig-a gene mutation assay, a powerful tool for evaluating in vivo genotoxicity, is based on flow cytometric enumeration of red blood cells (RBCs), which are deficient in glycosylphosphatidylinositol anchored proteins caused by mutation(s) in the Pig-a gene. Various approaches for measuring cells with mutated Pig-a gene have been developed. The Pig-a assay targeting concentrated reticulocytes – the PIGRET assay – has the potential to detect genotoxicity in early stages of the study. To verify the potential and usefulness of the PIGRET assay for short-term testing, we conducted a joint research with the Mammalian Mutagenicity Study (MMS) Group of the Japanese Environmental Mutagen Society. As part of this study, we evaluated the genotoxicity of a single oral administration of acrylamide (AA) at 25, 50, 100, 137.5, and 175mg/kg using the PIGRET and Pig-a assays targeting RBCs (RBC Pig-a assay) at 7, 14, and 28 days after dosing. Toxic effects induced by AA, such as hind limb weak-paralysis, reduction of body weight gain, and reticulocytosis, were observed in AA-treated groups. However, we detected no significant increases in Pig-a mutant frequencies using either the PIGRET or RBC Pig-a assay. Therefore, we concluded that the genotoxicity of AA could not be detected by these assays under our experimental conditions.

Evaluation of in vivo mutagenicity of isopropyl methanesulfonate by RBC Pig-a and PIGRET assays

A comparison between the original red blood cell (RBC) Pig-a assay, which measures Pig-a mutant cells in RBCs, and the PIGRET assay, which uses reticulocytes, was conducted using the in vivo mutagenesis assay with isopropyl methanesulfonate (iPMS) as a part of a collaborative study by the Mammalian Mutagenicity Study Group in the Japanese Environmental Mutagen Society. Three dose levels of iPMS (50, 100, and 200mg/kg) were administered once intraperitoneally to 8-week-old male Crl:CD(SD) rats, and peripheral blood was sampled at 0 (1 day before dosing), and 1, 2, and 4 weeks after dosing with iPMS. As a result, a time-dependent increase in the mutant frequency of Pig-a mutant RBCs was observed in the RBC Pig-a assay, and a statistically significant increase was observed from 2 weeks after dosing. In the PIGRET assay, on the other hand, a statistically significant increase in Pig-a mutant frequency was obtained from 1 week after dosing at all dose levels, and the Pig-a mutant frequency at the highest dose level had already reached a plateau on week 1. The maximum Pig-a mutant frequency induced by a single treatment with iPMS at 200mg/kg in the PIGRET assay was approximately two times higher than that in the RBC Pig-a assay. These results indicate that the PIGRET assay can detect Pig-a mutants much earlier and with a higher value in Pig-a mutant frequency compared with the original RBC Pig-a assay, and it can enable judgement of mutagenicity of iPMS within 1 week after a single dose.

PIGRET assay can detect mutagenicity of ethyl methanesulfonate much earlier than RBC Pig-a assay

The comparison between the original red blood cell (RBC) Pig-a assay, which measures Pig-a mutant RBCs, and the PIGRET assay, which uses reticulocytes, was conducted using in vivo mutagenesis by ethyl methanesulfonate (EMS) as a part of a collaborative study by the Mammalian Mutagenicity Study Group in the Japanese Environmental Mutagen Society. Three dose levels of EMS (180, 360, and 720mg/kg) were administered once by oral gavage to 8-week-old male Crl:CD(SD) rats, and peripheral blood was sampled at 0 (1 day before dosing), 1, 2, and 4 weeks after dosing with EMS. As a result, a statistically significant increase in the mutant frequency of the Pig-a gene was observed from 2 weeks after dosing and a higher value was obtained on week 4 at the highest dose only in the RBC Pig-a assay. In the PIGRET assay, on the other hand, a statistically significant increase in Pig-a mutant frequency was obtained at the highest dose from 1 week after dosing, and it decreased on weeks 2 and 4 compared to the value at week 1. The Pig-a mutant frequency appeared to reach a plateau 1 week after dosing in the PIGRET assay and it might continue to increase even after week 4 in the RBC Pig-a assay. These results indicate that the PIGRET assay can detect Pig-a mutants much earlier than the original RBC Pig-a assay, and it can enable judgement of mutagenicity of EMS within 1 week after a single dosing.

Persistence of red blood cells with <i>Pig-a</i> mutation in <i>p53</i> knockout mice exposed to X-irradiation

The Pig-a mutation assay is becoming one of the major experimental procedures used to assess in vivo genotoxicity. The assay allows simple in vivo analysis and enables gene mutations in the hematopoietic system to be measured using high throughput flow cytometry. Previously, we demonstrated that X-irradiation increased the Pig-a mutant frequencies in red blood cells (RBCs) of mice in a radiation dose-dependent manner. In this study, to understand how RBCs with Pig-a mutation induced by X-irradiation persist, we compared Pig-a mutant frequencies between irradiated C57BL/6J (p53(+/+)) mice and irradiated p53 homozygous knockout (p53(-/-)) mice by using the RBC Pig-a assay. After the peak in radiation-induced Pig-a mutant frequencies, a gradual decrease in mutant frequencies in irradiated p53(-/-) mice was observed, while irradiated p53(+/+) mice had a rapid decrease, which suggests that RBCs with Pig-a mutation are eliminated normally in irradiated p53(+/+) mice but not in irradiated p53(-/-) mice due to lack of p53 function. In addition, we also found that the p53 function affected the regulation of Pig-a mutagenesis in aging mice. Our results suggest that p53 function, distinct types of mutation, and the life span of RBCs play key roles in the persistence of Pig-a mutation in the hematopoietic system of RBCs after irradiation.

Monitoring humans for somatic mutation in the endogenous pig‐a gene using red blood cells

The endogenous X-linked PIG-A gene is involved in the synthesis of glycosyl phosphatidyl inositol (GPI) anchors that tether specific protein markers to the exterior of mammalian cell cytoplasmic membranes. Earlier studies in rodent models indicate that Pig-a mutant red blood cells (RBCs) can be induced in animals treated with genotoxic agents, and that flow cytometry can be used to identify rare RBCs deficient in the GPI-anchored protein, CD59, as a marker of Pig-a gene mutation. We investigated if a similar approach could be used for detecting gene mutation in humans. We first determined the frequency of spontaneous CD59-deficient RBCs (presumed PIG-A mutants) in 97 self-identified healthy volunteers. For most subjects, the frequency of CD59-deficient RBCs was low (average of 5.1 ± 4.9 × 10(-6) ; median of 3.8 × 10(-6) and mutant frequency less than 8 × 10(-6) for 75% of subjects), with a statistically significant difference in median mutant frequencies between males and females. PIG-A RBC mutant frequency displayed poor correlation with the age and no correlation with the smoking status of the subjects. Also, two individuals had markedly increased CD59-deficient RBC frequencies of ∼300 × 10(-6) and ∼100 × 10(-6) . We then monitored PIG-A mutation in 10 newly diagnosed cancer patients undergoing chemotherapy with known genotoxic drugs. The frequency of CD59-deficient RBCs in the blood of the patients was measured before the start of chemotherapy and three times over a period of ∼6 months while on/after chemotherapy. Responses were generally weak, most observations being less than the median mutant frequency for both males and females; the greatest response was an approximate three-fold increase in the frequency of CD59-deficient RBCs in one patient treated with a combination of cisplatin and etoposide. These results suggest that the RBC PIG-A assay can be adopted to measuring somatic cell mutation in humans. Further research is necessary to determine the assay's sensitivity in detecting mutations induced by genotoxic agents acting via different mechanisms.