Increases In Pig-a Mutant Frequency Research Articles

PIG-A gene mutation as a genotoxicity biomarker in polycyclic aromatic hydrocarbon-exposed barbecue workers

BackgroundThe PIG-A gene mutation assay is a valuable tool for measuring in vivo gene mutations in blood cells. The human PIG-A assay, used as a potential genotoxicity biomarker, is minimally invasive, sensitive, and cost-efficient; however, the relationship between carcinogen exposure and PIG-A mutations is not well understood.MethodsWe investigated the genotoxic effect of red blood cells using PIG-A assay and lymphocyte cytokinesis-block micronucleus test in barbecue restaurant workers (N = 70) exposed to polycyclic aromatic hydrocarbons (PAHs) and self-identified healthy control subjects (N = 56). Urinary PAH metabolites were measured to evaluate internal exposure levels.ResultsMultivariate Poisson regression showed that the PAH-exposed workers exhibited significantly higher PIG-A mutant frequency (MF) (8.04 ± 6.81 × 10− 6) than did the controls (5.56 ± 5.26 × 10− 6) (RR = 0.707, 95% CI: 0.615–0.812, P < 0.001). These results indicate that PAH exposure is a risk factor for elevated PIG-A MF. The frequencies of micronuclei (MN) and nuclear buds (NBUD) in the PAH-exposed workers (MN: 3.06 ± 2.07 ‰, NBUD: 1.38 ± 1.02 ‰) were also significantly higher than in the controls (MN: 1.46 ± 0.64 ‰, P < 0.001; NBUD: 0.70 ± 0.60 ‰, P < 0.001). Additionally, PIG-A MFs showed better associations with several urinary hydroxylated PAH metabolites (P2-OH-Flu = 0.032, r2-OH-Flu = 0. 268; P2-OH-Phe = 0.022, r2-OH-Phe = 0.286; P3-OH-Phe = 0.0312, r3-OH-Phe = 0.270; P4-OH-Phe = 0.018, r4-OH-Phe = 0.296), while the increase in MN, NPB, and NBUD frequencies was not associated with any OH-PAH metabolites; and high-PAH-exposed workers showed the highest PIG-A MFs. Furthermore, there was a significant association between PIG-A MF and PAH exposure levels (Chi-square test for trend, P = 0.006).ConclusionsOur results indicate that an increase in PIG-A MF in barbecue workers could reflect the response to PAH exposure, providing evidence of its potential as a genotoxicity biomarker in human risk assessment.

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 in vivo gene mutation with etoposide using Pig-a and PIGRET assays

The Pig-a assay detects the expression of the endogenous phosphatidylinositol glycan anchor biosynthesis, class A gene (Pig-a) as a reporter of mutations and shows promise for detecting mutations in vivo. This assay requires two to four weeks to detect mutations in erythrocytes after animals are administered a single dose of test compound. In contrast, the more recently developed PIGRET assay using reticulocytes detects mutation sensitively one week after dosing, which is an advantage for conducting short-term genotoxicity studies in vivo. As part of the Japanese Environmental Mutagen Society/Mammalian Mutagenicity Study Group collaborative study, we conducted Pig-a and PIGRET assays to detect Pig-a mutations in rats treated with etoposide. The DNA topoisomerase II-inhibitor, etoposide, causes DNA cleavage and subsequent protein-linked DNA double-strand breaks, induces chromosomal aberrations and micronuclei, and is classified as 2A (probably carcinogenic to humans) by IARC. Etoposide was intravenously given once to 7-week old SD rats at doses of 0, 5, 10, and 20mg/kg. Severe myelosuppression was induced 2days after dosing in all dosing groups. We conducted Pig-a mutant analysis in Pig-a and PIGRET assays 1, 2, and 4 weeks after dosing. Neither Pig-a nor PIGRET assays showed any statistically significant increases in Pig-a mutant frequency in any of the dose groups at any of the sampling times. It was concluded that etoposide was judged negative in Pig-a and PIGRET assays of bone marrow cells, and the results of the two assays showed hardly any differences.

Report on the rat Pig-a assay using an anti-rat erythroid marker HIS49 antibody in a single dose study of 1,2-dimethylhydrazine

As part of a collaborative study in the Mammalian Mutagenicity Study (MMS) Group of the Japanese Environmental Mutagen Society (JEMS), we investigated the in vivo genotoxicity profile of 1,2-dimethylhydrazine (DMH) using a Pig-a assay in total red blood cells (RBC Pig-a assay) or a reticulocyte Pig-a assay (PIGRET assay). We also assessed the genotoxic potential of DMH using both a bone marrow micronucleus test and a liver comet assay as follow-up studies. Single administration of 25, 50, 100mg/kg DMH to male rats did not show time- or dose-related increases in Pig-a mutant frequency (MF) in either the RBC Pig-a or PIGRET assays up to 4 weeks after treatment. The bone marrow micronucleus test under the same dose levels was judged positive, while the liver comet assay was judged inconclusive due to the high number of hedgehogs. Re-evaluation of the rat liver comet assay at lower dose levels (4, 10, and 25mg/kg DMH) showed a dose-related increase in%DNA in tail. Taken together, DMH showed a positive response in both the bone marrow micronucleus test and liver comet assay, while the increases in Pig-a MF in both the RBC Pig-a and PIGRET assays could hardly be detected after single dosing. These results suggest that DMH provides different genotoxicity outcomes depending on the endpoint following acute in vivo dosing.

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.

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.

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.

Interlaboratory trial of the rat Pig-a mutation assay using an erythroid marker HIS49 antibody

The peripheral blood Pig-a assay has shown promise as a tool for evaluating in vivo mutagenicity. In this study five laboratories participated in a collaborative trial that evaluated the transferability and reproducibility of a rat Pig-a assay that uses a HIS49 antibody reacts with an antigen found on erythrocytes and erythroid progenitors. In preliminary work, flow cytometry methods were established that enabled all laboratories to detect CD59-negative erythrocyte frequencies (Pig-a mutant frequencies) of <10×10−6 in control rats. Four of the laboratories (the in-life labs) then treated male rats with a single oral dose of N-nitroso-N-ethylurea, 7,12-dimethylbenz[a]anthracene (DMBA), or 4-nitroquinoline-1-oxide (4NQO). Blood samples were collected up to 4 weeks after the treatments and analyzed by flow cytometry for the frequency of CD59-negative cells among total red blood cells (RBCs; RBC Pig-a assay). RBC Pig-a assays were conducted in the four in-life laboratories, plus a fifth laboratory that received blood samples from the other laboratories. In addition, three of the five laboratories performed a Pig-a assay on reticulocytes (RETs; PIGRET assay), using blood from the rats treated with DMBA and 4NQO. The four in-life laboratories detected consistent, time- and dose-related increases in RBC Pig-a mutant frequency (MF) for all three test articles. Furthermore, comparable results were obtained in the fifth laboratory that received blood samples from other laboratories. The three laboratories conducting the PIGRET assay also detected consistent, time- and dose-related increases in Pig-a MF, with the RET MFs increasing more rapidly with time than RBC MFs. These results indicate that rat Pig-a assays using a HIS49 antibody were transferable between laboratories and that data generated by the assays were reproducible. The findings also suggest that the PIGRET assay may detect the in vivo mutagenicity of test compounds earlier than the RBC Pig-a assay.

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

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.

The micronucleus assay with 1,3-propane sultone

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.