Pig-a Assay Research Articles

Evaluation of PIG-A-mutated granulocytes and ex-vivo binucleated micronucleated lymphocytes frequencies after breast cancer radiotherapy in humans.

Although the PIG-A gene mutation frequency (MF) is considered a good proxy to evaluate the somatic MF in animals, evidence remains scarce in humans. In this study, a granulocyte PIG-A-mutant assay was evaluated in patients undergoing radiation therapy (RT) for breast cancer. Breast cancer patients undergoing adjuvant RT were prospectively enrolled. RT involved the whole breast, with (WBNRT) or without (WBRT) nodal area irradiation. Blood samples were obtained from participants before (T0) RT, and T1, T2, and T3 samples were collected 3 weeks after the initiation of RT, at the end of RT, and at least 10 weeks after RT discontinuation, respectively. The MF was assessed using a flow cytometry protocol identifying PIG-A-mutant granulocytes. Cytokinesis-blocked micronucleated lymphocyte (CBML) frequencies were also evaluated. Thirty patients were included, and five of them had received chemotherapy prior to RT. The mean (±SD) PIG-A MFs were 7.7 (±12.1) per million at T0, 5.2 (±8.6) at T1, 6.4 (±8.0) at T2 and 3.8 (±36.0) at T3. No statistically significant increases were observed between the PIG-A MF at T0 and the MFs at other times. RT significantly increased the CBML frequencies: 7.9 ‰ (±3.1‰) versus 33.6‰ (±17.2‰) (p < .0001). By multivariate analysis, the CBML frequency was correlated with age at RT initiation (p = .043) and irradiation volume at RT discontinuation (p = .0001) but not with chemotherapy. RT for breast cancer therapy failed to induce an increase in the PIG-A MF. The PIG-A assay in humans needs further evaluation, in various genotoxic exposures and including various circulating human cells.

Molecular analysis of GPI-anchor biosynthesis pathway genes in rat strains used for the Pig-a gene mutation assay

Recent studies support the assumption that mutation of the X-linked Pig-a gene is most likely responsible for the mutant phenotype of the cells deficient in glycosylphosphatidylinositol (GPI)-anchored proteins quantified in the rodent Pig-a gene mutation assay. In humans, however, mutations in both alleles of one of the 30 other genes involved in GPI-anchor synthesis, e.g., PIG-L and PIG-O, cause reduced expression of surface GPI-anchored proteins. Here, we investigated the possibility that the loss of the GPI-anchor detected by the rat Pig-a assay also could be caused by mutation in other GPI-biosynthesis genes. 31 samples were obtained from 8 inbred and outbred rat strains commonly used for genetic toxicology assays. In order to investigate possible sources of variation in the Pig-a assay, variant DNA sequences were evaluated in Cd59 and 24 GPI-biosynthesis genes. In some genes, such as Pig-n and Pig-u, homozygous variations occurred in all animals, suggesting that these variations are due to deviations in the reference genome. Heterozygous Pig-s, Pig-w, Pig-o, Pig-c, Pgap1, Pgap2, Pig-k and Pig-t variations were found, however, indicating that these genes could serve as targets for mutation in the assay. Protein alignment for these altered genes was conducted with possible human, mouse and rat phenotypic mutants from the literature; this analysis demonstrated that many of the variations that we detected were in non-conserved sequences and that no phenotypes for any of these variants could be inferred from known mutants from the literature. All heterozygous variants were in outbred rats. Overall, the findings of this study cannot totally rule out the possibility that mutations in GPI-biosynthesis genes other than Pig-a are detected in the Pig-a assay, but suggest that if it occurs, it must occur only rarely and therefore mutations in genes other than Pig-a have little impact on rat-based experiments.

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.

Pig-a gene mutation assay study design: critical assessment of 3- versus 28-day repeat-dose treatment schedules.

The in vivo Pig-a assay is being used in safety studies to evaluate the potential of chemicals to induce somatic cell gene mutations. Ongoing work is aimed at developing an Organisation for Economic Cooperation and Development (OECD) test guideline to support routine use for regulatory purposes (OECD project number 4.93). Among the details that will need to be articulated in an eventual guideline are recommended treatment and harvest schedules. With this in mind, experiments reported herein were performed with Wistar Han rats exposed to aristolochic acid I (AA), 1,3-propane sultone, chlorambucil, thiotepa or melphalan using each of two commonly used treatment schedules: 3 or 28 consecutive days. In the case of the 3-day studies, blood was collected for Pig-a analysis on days 15 or 16 and 29 or 30. For the 28-day studies blood was collected on day 29 or 30. The effect of treatment on mutant reticulocytes and mutant erythrocytes was evaluated with parametric pair-wise tests. While each of the five mutagens increased mutant phenotype cell frequencies irrespective of study design, statistical significance was consistently achieved at lower dose levels when the 28-day format was used (e.g. 2.75 vs 20 mg/kg/bw for AA). To more thoroughly investigate the dose-response relationships, benchmark dose (BMD) analyses were performed with PROAST software. These results corroborate the pair-wise testing results in that lower BMD values were obtained with the 28-day design. Finally, mutagenic potency, as measured by BMD analyses, most consistently correlated with the mutagens' tumorigenic dose 50 values when the lengthier treatment schedule was used. Collectively, these results suggest that both 3- and 28-day treatment schedules have merit in hazard identification-type studies. That being said, for the purpose of regulatory safety assessments, there are clear advantages to study designs that utilise protracted exposures.

PIG-A gene mutation as a genotoxicity biomarker in human population studies: An investigation in lead-exposed workers.

The rodent Pig-a gene mutation assay has demonstrated remarkable sensitivity in identifying in vivo mutagens, while much less is known about the value of the human PIG-A assay for risk assessment. To obtain more evidence of its potential as a predictive biomarker for carcinogen exposure, we investigated PIG-A mutant frequencies (MFs), along with performing the Comet assay and micronucleus (MN) test, in 267 workers occupationally exposed to lead. Multivariate Poisson regression showed that total red blood cell PIG-A MFs were significantly higher in lead-exposed workers (10.90 ± 10.7 × 10-6 ) than in a general population that we studied previously (5.25 ± 3.6 × 10-6 ) (p < .0001). In contrast, there was no increase in lymphocyte MN frequency or in DNA damage as measured by percentage comet tail intensity in whole blood cells. Current year worker blood lead levels (BLL), an exposure biomarker, were elevated (232.6 ± 104.6 μg/L, median: 225.4 μg/L); a cumulative blood lead index (CBLI) also was calculated based on a combination of current and historical worker BLL data. Chi-square testing indicated that PIG-A MFs were significantly related to CBLI (p = .0249), but independent of current year BLL (p = .4276). However, % comet tail intensity and MN frequencies were better associated with current year BLL than CBLI. This study indicates that the PIG-A assay could serve as biomarker to detect the genotoxic effects of lead exposure and demonstrates that a battery of genotoxicity biomarkers having mechanistic complementarity may be useful for comprehensively monitoring human carcinogenic risk.

Testing of acetaminophen in support of the international multilaboratory in vivo rat Pig-a assay validation trial.

Acetaminophen, a nonmutagenic compound as previously concluded from bacteria, in vitro mammalian cell, and in vivo transgenic rat assays, presented a good profile as a nonmutagenic reference compound for use in the international multilaboratory Pig‐a assay validation. Acetaminophen was administered at 250, 500, 1,000, and 2,000 mg·kg−1·day−1 to male Sprague Dawley rats once daily in 3 studies (3 days, 2 weeks, and 1 month with a 1‐month recovery group). The 3‐Day and 1‐Month Studies included assessments of the micronucleus endpoint in peripheral blood erythrocytes and the comet endpoint in liver cells and peripheral blood cells in addition to the Pig‐a assay; appropriate positive controls were included for each assay. Within these studies, potential toxicity of acetaminophen was evaluated and confirmed by inclusion of liver damage biomarkers and histopathology. Blood was sampled pre‐treatment and at multiple time points up to Day 57. Pig‐a mutant frequencies were determined in total red blood cells (RBCs) and reticulocytes (RETs) as CD59‐negative RBC and CD59‐negative RET frequencies, respectively. No increases in DNA damage as indicated through Pig‐a, micronucleus, or comet endpoints were seen in treated rats. All positive controls responded as appropriate. Data from this series of studies demonstrate that acetaminophen is not mutagenic in the rat Pig‐a model. These data are consistent with multiple studies in other nonclinical models, which have shown that acetaminophen is not mutagenic. At 1,000 mg·kg−1·day−1, Cmax values of acetaminophen on Day 28 were 153,600 ng/ml and 131,500 ng/ml after single and repeat dosing, respectively, which were multiples over that of clinical therapeutic exposures (2.6–6.1 fold for single doses of 4,000 mg and 1,000 mg, respectively, and 11.5 fold for multiple dose of 4,000 mg) (FDA 2002). Data generated were of high quality and valid for contribution to the international multilaboratory validation of the in vivo Rat Pig‐a Mutation Assay.

Intra- and inter-laboratory reproducibility of the rat blood Pig-a gene mutation assay.

The in vivo Pig-a assay is being used in safety studies to evaluate the potential of chemicals to induce somatic cell gene mutations. Ongoing work is aimed at developing an Organization for Economic Cooperation and Development (OECD) test guideline to support routine use for regulatory purposes (OECD project number 4.93). Among the requirements for OECD approval are demonstrations of assay reliability, including reproducibility within and among laboratories. Experiments reported herein address the reproducibility of the rat blood Pig-a assay using the reference mutagens chlorambucil and melphalan. These agents were evaluated for their ability to induce Pig-a mutant erythrocytes in three separate studies conducted across two laboratories. Each of the studies utilized a common treatment schedule: 28 consecutive days of exposure via oral gavage. Whereas one laboratory studied Crl:CD(SD) rats, the other laboratory used Wistar Han rats. One or two days after cessation of treatment blood samples were collected for mutant reticulocyte and mutant erythrocyte measurements that were accomplished with the same analytical technique whereby samples were depleted of wildtype erythrocytes via immunomagnetic separation followed by flow cytometric enumeration of mutant phenotype cells (MutaFlow®). Dunnett's test results showed similar qualitative outcomes within and between laboratories, that is, each chemical and each study demonstrated statistically significant, dose-related increases in mutant reticulocyte and erythrocyte frequencies. Benchmark dose analysis (PROAST software) provided a means to quantitatively analyze the results, and the relatively tight, overlapping benchmark dose confidence intervals observed for each of the two chemicals indicate that within and between laboratory reproducibility of the Pig-a assay are high, adding further support for the development of an OECD test guideline.

Dextran sulfate sodium mouse model of inflammatory bowel disease evaluated for systemic genotoxicity via blood micronucleus and Pig-a gene mutation assays

Inflammatory bowel disease (IBD) is an important risk factor for gastrointestinal cancers. Inflammation and other carcinogenesis-related effects at distal, tissue-specific sites require further study. In order to better understand if systemic genotoxicity is associated with IBD, we exposed mice to dextran sulfate sodium salt (DSS) and measured the incidence of micronucleated cells (MN) and Pig-a mutant phenotype cells in blood erythrocyte populations. In one study, 8-week-old male CD-1 mice were exposed to 0, 1, 2, 3 or 4% w/v DSS in drinking water. The 4-week in-life period was divided into four 1-week intervals-alternately on then off DSS treatment. Low volume blood samples were collected for MN analysis at the end of each week, and cardiac blood samples were collected at the end of the 4-week period for Pig-a analyses. The two highest doses of DSS were observed to induce significant increases in reticulocyte frequencies. Even so, no statistically significant treatment-related effects on the genotoxicity biomarkers were evident. While one high-dose mouse showed modestly elevated MN frequencies during the DSS treatment cycles, it also exhibited exceptionally high reticulocyte frequencies (e.g. 18.7% at the end of the second DSS cycle). In a second study, mice were treated with 0 or 4% DSS for 9-18 consecutive days. Exposure was continued until rectal bleeding or morbidity was evident, at which point the treatment was terminated and blood was collected for MN analysis. The Pig-a assay was conducted on samples collected 29 days after the start of treatment. The initial blood specimens showed highly elevated reticulocyte frequencies in DSS-exposed mice (mean ± SEM = 1.75 ± 0.10% vs. 13.04 ± 3.66% for 0 vs. 4% mice, respectively). Statistical analyses showed no treatment-related effect on MN or Pig-a mutant frequencies. Even so, the incidence of MN versus reticulocytes in the DSS-exposed mice were positively correlated (linear fit R2 = 0.657, P = 0.0044). Collectively, these results suggest that in the case of the DSS CD-1 mouse model, systemic effects include stress erythropoiesis but not remarkable genotoxicity. To the extent MN may have been slightly elevated in a minority of individual mice, these effects appear to be secondary, likely attributable to stimulated erythropoiesis.

The potential application of human PIG-A assay on azathioprine-treated inflammatory bowel disease patients.

The rodent Pig-a assay has been used extensively as a potential regulatory assay for evaluating the in vivo mutagenicity of test substances. Although the assay can be conducted in different mammalian species, there have been only a few reports describing its use in humans, and rarely in genotoxicant-exposed human populations. In this study, PIG-A mutation frequencies (MFs) were evaluated in 36 azathioprine (AZA; human carcinogen)-treated inflammatory bowel disease (IBD) patients and 36 healthy volunteers. IBD patients exhibited a slight but statistically higher MF (6.10 ± 4.44 × 10-6 ) than healthy volunteers (4.97 ± 2.74 × 10-6 ) (P = 0.0489). The estimated relative risk for the exposed patients was 1.22 which indicated that AZA is a risk factor for inducing PIG-A mutation. However, the PIG-A MF showed no associations with AZA treatment duration or total AZA exposure. In addition, we performed the cytokinesis-block micronucleus test on the same samples. The frequencies of micronuclei (MN) and nuclear buds (NBUD) in IBD patients (MN: 4.70 ± 2.86‰; NBUD: 1.89 ± 0.95‰) were significantly higher than in healthy volunteers (MN: 1.47 ± 0.77‰, P < 0.001; NBUD: 0.90 ± 0.58‰, P = 0.004). MN frequency also had significant correlations with AZA treatment duration (P = 0.011) and total AZA exposure (P = 0.018). Our findings indicate that AZA-treated IBD patients have only a marginally significant increase in PIG-A MF; in contrast, a much stronger AZA-associated increase in genotoxicity was detected with the lymphocyte MN assay. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.

Assessment of Pig-a, Micronucleus, and Comet Assay Endpoints in Tg.RasH2 Mice Carcinogenicity Study of Aristolochic Acid I.

A newly developed in vivo Pig-a gene mutation assay displays great potential for integration into genotoxicity tests. To obtain more evidence for application of the Pig-a assay, we integrated this assay, micronucleus test in peripheral blood (MN-pb test) and bone marrow (MN-bm test), as well as a Comet assay into a transgenic RasH2 mice carcinogenicity study. Fourteen male RasH2 mice and five wild-type (WT) mice were treated with a strong mutagen aristolochic acid I at a dose of 5 mg/kg/day for 4 consecutive weeks. Mice recovered in 5 weeks. Peripheral bloods were collected for Pig-a assay, MN-pb test, and Comet assay at several time points, while bone marrow and target organs were harvested for the MN-bm test and pathological diagnosis after mice were euthanized. Finally, 13 of the 14 RasH2 mice developed squamous cell carcinomas in the forestomach, while there were no carcinomas in the WT mice. Pig-a mutant frequencies (MFs) consecutively increased throughout the study to a maximum value of approximately 63-fold more than background. These frequencies were relative to the incidence, size, and malignant degree of tumors. Micronucleated reticulocytes increased from Day 1 to Day 49, before returning to background levels. No positive responses were observed in either the MN-bm test or the Comet assay. Results suggested that, when compared with the other two tests, the Pig-a assay persistently contributed to sustaining MFs, enhanced detection sensitivity due to the accumulation of Pig-a mutations, and demonstrated better predictability for tumorigenicity. Environ. Mol. Mutagen. 61:266-275, 2020. © 2019 Wiley Periodicals, Inc.

Caffeic Acid Genotoxicity: Correlation of the Pig-a Assay with Regulatory Genetic Toxicology In Vivo Endpoints.

Caffeic acid is found in variety of fruits and vegetables. It is considered as possible human carcinogen (Group 2B). It is negative in Ames and mouse micronucleus (MN), but positive in mouse lymphoma and chromosomal aberration assays. The objective of this study was to evaluate the in vivo genotoxicity of caffeic acid using three different endpoints: in vivo MN, Pig-a, and comet assay. Two sets of six rats per group were administered vehicle (0.5% hydroxypropyl methylcellulose), 500, 1,000, or 2,000 mg/kg/day of caffeic acid for three consecutive days via oral gavage. One set of animals was used for the Pig-a and MN assay and the other set was used for the comet assay. N-Ethyl N-Nitrosourea was used as positive control for the Pig-a and MN assay, and ethyl methanesulfonate for the comet assay. From one set of animals, peripheral blood was collected on Days -1, 14, and 30 for the Pig-a assay and on Day 4 for the MN assay. The other set of animals was euthanized 3 hr after the last dose; liver and blood were collected for the comet assay. A statistically significant increase in the MN frequency was observed at 2,000 mg/kg/day. No increase in the red blood cells (RBCCD59- ) or reticulocytes (RETCD59- ) Pig-a mutant frequencies was observed on Days 14 or 30. No increase in DNA strand breaks was observed in the peripheral blood or liver in the comet assay. Environ. Mol. Mutagen. 2019. © 2019 Wiley Periodicals, Inc.

3Rs friendly study designs facilitate rat liver and blood micronucleus assays and Pig-a gene mutation assessments: Proof-of-concept with 13 reference chemicals.

Regulatory guidance documents stress the value of assessing the most appropriate endpoints in multiple tissues when evaluating the in vivo genotoxic potential of chemicals. However, conducting several independent studies to evaluate multiple endpoints and/or tissue compartments is resource intensive. Furthermore, when dependent on visual detection, conventional approaches for scoring genotoxicity endpoints can be slow, tedious, and less objective than the ideal. To address these issues with current practices we attempted to (1) devise resource sparing treatment and harvest schedules that are compatible with liver and blood micronucleus endpoints, as well as the Pig-a gene mutation assay, and (2) utilize flow cytometry-based methods to score each of these genotoxicity biomarkers. Proof-of-principle experiments were performed with 4-week-old male and female Crl:CD(SD) rats exposed to aristolochic acids I/II, benzo[a]pyrene, cisplatin, cyclophosphamide, diethylnitrosamine, 1,2-dimethylhydrazine, dimethylnitrosamine, 2,6-dinitrotoluene, hydroxyurea, melphalan, temozolomide, quinoline, or vinblastine. These 13 chemicals were each tested in two treatment regimens: one 3-day exposure cycle, and three 3-day exposure cycles. Each exposure, blood collection, and liver harvest was accomplished during a standard Monday-Friday workweek. Key findings are that even these well-studied, relatively potent genotoxicants were not active in both tissues and all assays (indeed only cisplatin was clearly positive in all three assays); and whereas the sensitivity of the Pig-a assay clearly benefitted from three versus one treatment cycle, micronucleus assays yielded qualitatively similar results across both study designs. Collectively, these results suggest it is possible to significantly reduce animal and other resource requirements while improving assessments of in vivo genotoxicity potential by simultaneously evaluating three endpoints and two important tissue compartments using fit-for-purpose study designs in conjunction with flow cytometric scoring approaches. Environ. Mol. Mutagen., 60:704-739, 2019. © 2019 Wiley Periodicals, Inc.

Integration of micronucleus, comet, and Pig-a gene mutation endpoints into rat 15-day repeat-treatment studies: Proof-of-principle with Auramine O

A series of genotoxicity assessments were conducted on male Sprague Dawley rats treated with Auramine O (AO) to establish a multiple-endpoint assay. The rat liver micronucleus assay, in combination with the comet assay, peripheral blood micronucleus assay, and erythrocyte Pig-a assay in the same experiment, comprehensively assess the genotoxicity of AO. Rats were orally exposed to 0, 100, 200, or 400 mg/kg/day AO for 15 consecutive days. The blood was sampled on Days -1 and 15 for the erythrocyte Pig-a assay and peripheral blood micronucleus assay. Livers were sampled on Day 15 for the liver micronucleus assay and comet assay. Based on the liver micronucleus assay and liver comet assay, AO induced a significant dose-related increase of micronucleated hepatocyte frequencies, and tail DNA percentages, respectively in the middle- and high-dose groups. On the blood micronucleus test and Pig-a assay, no significant increases were observed for the micronucleated reticulocyte frequencies, mutant erythrocyte frequencies (RBCCD59−) or mutant reticulocyte frequencies (RETCD59−) at any of the time points studied. In conclusion, using a multiple-endpoint genotoxicity assay method can reduce the number of experimental animals, boost the efficiency of the experiment, and improve the accuracy of investigations of genotoxicity.

In vivo genotoxicity testing strategies: Report from the 7th International workshop on genotoxicity testing (IWGT)

The working group reached complete or majority agreement on many issues.Results from TGR and in vivo comet assays for 91 chemicals showed they have similar ability to detect in vivo genotoxicity per se with bacterial mutagens and Ames-positive carcinogens.TGR and comet assay results were not significantly different when compared with IARC Group 1, 2 A, and unclassified carcinogens. There were significantly more comet assay positive responses for Group 2B chemicals, and for IARC classified and unclassified carcinogens combined, which may be expected since mutation is a sub-set of genotoxicity.A liver comet assay combined with the bone marrow/blood micronucleus (MNviv) test would detect in vivo genotoxins that do not exhibit tissue-specific or site-of-contact effects, and is appropriate for routine in vivo genotoxicity testing.Generally for orally administered substances, a comet assay at only one site-of-contact GI tract tissue (stomach or duodenum/jejunum) is required.In MNviv tests, evidence of target tissue exposure can be obtained in a number of different ways, as recommended by ICH S2(R1) and EFSA (Hardy et al., 2017).Except for special cases the i.p. route is inappropriate for in vivo testing; for risk evaluations more weight should be given to data from a physiologically relevant administration route.The liver MN test is sufficiently validated for the development of an OECD guideline. However, the impact of dosing animals >6 weeks of age needs to be evaluated. The GI tract MN test shows promise but needs more validation for an OECD guideline.The Pig-a assay detects systemically available mutagens and is a valuable follow-up to in vitro positive results. A new freeze-thaw protocol provides more flexibility. Mutant reticulocyte and erythrocyte frequencies should both be determined. Preliminary data are available for the Pig-a assay in male rat germ cells which require validation including germ cell DNA mutation origin.

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.

Induction of in vivo Pig-a gene mutation but not micronuclei by 5-(2-chloroethyl)-2'-deoxyuridine, an antiviral pyrimidine nucleoside analogue.

5-(2-Chloroethyl)-2'-deoxyuridine (CEDU) was developed as an antiviral drug. It has been studied in a number of in vitro and in vivo genotoxicity assays and is considered an unusual nucleoside analogue owing to its potent mutagenic potential, with little to no measurable clastogenic activity. Given this atypical profile, CEDU represented an interesting compound for evaluating the in vivo Pig-a gene mutation assay, a test that is undergoing extensive validation for regulatory safety applications. The current report describes two studies with 7-week-old male Wistar Han rats, one that exposed animals to several dose levels of CEDU for 5 consecutive days, the other for 28 consecutive days. Blood samples were collected at several time points and analysed for Pig-a mutant cell frequencies via flow cytometry. These Pig-a analyses were accompanied by micronucleated reticulocyte (MN-RET) measurements performed with blood samples collected 1 day after cessation of treatment. Both studies showed robust CEDU dose-related increases in Pig-a mutant reticulocytes and mutant erythrocytes. Conversely, neither experiment showed evidence of a CEDU-related MN-RET-inducing effect. These rat haematopoietic cell results were in good agreement with those of earlier mouse studies where in vivo mutagenesis was observed, without clastogenicity/aneuploidy. Taken together, these data add further support to the concept that the Pig-a assay represents an important complement to the widely used in vivo micronucleus assay, as it expands the range of important DNA lesions that can be detected in short-term as well as protracted exposure study designs.

Application of the in vivo Pig-a gene mutation assay to test the potential genotoxicity of p-phenylenediamine

Currently, it remains controversial whether p-phenylenediamine (PPD) is genotoxic. In this study, we evaluated the potential genotoxicity of PPD using the newly-developed Pig-a gene mutation assay. The results of three classical genetic toxicity tests (bacterial reverse mutation assay, mammalian cell chromosomal aberration test, and mammalian erythrocyte micronucleus test) are all positive, suggesting that PPD is potentially genotoxic. In Pig-a assay, Sprague-Dawley rats are orally administered with PPD for 28 consecutive days at three doses (12.5, 25, and 50 mg/kg/day). Our result shows that PPD (25 and 50 mg/kg/day) dose-dependently increases RETCD59− value over controls on Day 8. RETCD59− keeps increasing to the maximum on Day 15 and then decreases until Day 29. PPD also dose-dependently increase RBCCD59− value on Day 15, which keeps elevating until Day 29. The time-course of RETCD59− and RBCCD59− induced by PPD are similar with that induced by N-ethyl-N-nitrosourea (ENU) treatment for 3 days. Our data suggests that PPD has potential genotoxic effects, and the Pig-a assay is sensitive to assess mutagenicity. However, further investigation of the changes of RETCD59− and RBCCD59− induced by hair dyes containing PPD should be detected by Pig-a assay in occupational exposure population to confirm the safety of PPD usage.

Evaluation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) mutagenicity using in vitro and in vivo Pig-a assays

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a genotoxic carcinogen found in tobacco and tobacco smoke. Several in vitro and in vivo assays have been used for evaluating the genotoxicity of tobacco smoke and tobacco smoke constituents like NNK, yet it is not clear which in vitro assays are most appropriate for extrapolating the in vitro responses of these test agents to animal models and humans. The Pig-a gene mutation assay can be performed in vitro, in laboratory animals, and in humans, a potential benefit in estimating in vivo responses from in vitro data. In the current study we used Pig-a as a reporter of gene mutation both in vitro, in L5178Y/Tk+/− cells, and in vivo, in Sprague-Dawley rats. NNK significantly increased Pig-a mutant frequency in L5178Y/Tk+/− cells, but only at concentrations of 100 μg/ml and greater, and only in the presence of S9 activation. Pig-a mutations in L5178Y/Tk+/− cells were detected in 80% of the NNK-induced mutants, with the predominate mutation being G→A transition; vehicle control mutants contained deletions. In the in vivo study, rats were exposed to NNK daily for 90 days by inhalation, a common route of exposure to NNK for humans. Although elevated mutant frequencies were detected, these responses were not clearly associated with NNK exposure, so that overall, the in vivo Pig-a assays were negative. Thus, while NNK induces mutations in the in vitro Pig-a assay, the in vivo Pig-a assay has limited ability to detect NNK mutagenicity under conditions relevant to NNK exposure in smokers.

Suitability of Long-Term Frozen Rat Blood Samples for the Interrogation of Pig-a Gene Mutation by Flow Cytometry.

The rodent blood Pig-a assay has been undergoing international validation for use as an in vivo hematopoietic cell gene mutation assay, and given the promising results an Organization for Economic Co-operation and Development (OECD) Test Guideline is currently under development. Enthusiasm for the assay stems in part from its alignment with 3Rs principles permitting combination with other genotoxicity endpoint(s) and integration into repeat-dose toxicology studies. One logistical requirement and experimental design limitation has been that blood samples required antibody labeling and flow cytometric analysis within one week of collection. In the current report, we describe the performance of freeze-thaw reagents that enable storage and subsequent labeling and analysis of rat blood samples for at least seven months. Data generated from three laboratories are presented that demonstrate rat erythrocyte recoveries in the range of 80-90%. Despite some loss of erythrocytes, Pearson coefficients and Bland-Altman analyses based on fresh blood vs. frozen/thawed matched pairs indicate that mutant cell and reticulocyte frequencies are not significantly affected, as the measurements are highly correlated and exhibit low bias. Collectively, these data support the effectiveness and suitability of a freeze-thaw procedure that endows the assay with several new advantageous characteristics that include: flexibility in scheduling personnel/instrumentation; reliability when shipping samples from in-life facilities to analytical sites; 3Rs-friendly, as blood from positive control animals can be stored frozen to serve as analytical controls; and ability to defer a decision to generate Pig-a data until more toxicological information becomes available on a test substance. Environ. Mol. Mutagen. 60:47-55, 2019. © 2018 Wiley Periodicals, Inc.

In vivo genotoxicity of 1,4-dioxane evaluated by liver and bone marrow micronucleus tests and Pig-a assay in rats

1,4-Dioxane, used widely as a solvent in the manufacture of chemicals and as a laboratory reagent, induced liver adenomas and carcinomas in mice and rats, and nasal tumors in rats in several long-term studies. 1,4-Dioxane has been reported to be non-genotoxic in vitro, and there is no clear conclusion concerning its in vivo genotoxicity in rodents. In the present study, we investigated the ability of 1,4-dioxane to induce micronuclei in the liver and bone marrow of rats. For the liver micronucleus test, we performed the juvenile animal method and two methods using partial hepatectomy (PH), dosing before PH or dosing after PH. We also evaluated the in vivo mutagenicity of 1,4-dioxane by Pig-a gene mutation assay using rat peripheral blood. As a result, all methods of liver micronucleus test showed an increase in the frequency of micronucleated hepatocytes by 1,4-dioxane. The dosing before PH, a suitable method for detecting structural chromosome aberration inducers, showed the clearest response for micronucleated hepatocytes induction among the three methods. This finding is consistent with a previous report that 1,4-dioxane induces mainly chromosome breakage in the liver. Negative results were obtained in the bone marrow micronucleus test and Pig-a gene mutation assay in our study. These results suggested that 1,4-dioxane is clastogenic in the liver but not genotoxic in the bone marrow of rats.