Takafumi Kimoto

National Institute of Health Sciences, Japan, Edo, Tōkyō, Japan

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Publications (12)37.55 Total impact

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    ABSTRACT: The in vivo Pig-a assay uses flow cytometry to measure phenotypic variants for antibody binding to cell surface glycosylphosphatidylinositol (GPI)-anchored proteins. There is good evidence suggesting that the absence of antibody binding is the result of a mutation in the endogenous X-linked Pig-a gene, which forms the rationale for the assay. Although the assay has been performed with several types of hematopoietic cells and in a variety of mammalian species, including humans, currently it is optimized only for measuring CD59-deficient (presumed Pig-a mutant) erythrocytes in the peripheral blood of rats. An expert workgroup formed by the International Workshop on Genotoxicity Testing considered the state of assay development and the potential of the assay for regulatory use. Consensus was reached on what is known about the Pig-a assay and how it should be conducted, and recommendations were made on additional data and refinements that would help to further enhance the assay for use in hazard identification and risk assessment.
    Mutation Research/Genetic Toxicology and Environmental Mutagenesis 01/2014; · 2.22 Impact Factor
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    ABSTRACT: The recently developed Pig-a mutation assay is based on flow cytometric enumeration of glycosylphosphatidylinositol (GPI) anchor-deficient red blood cells caused by a forward mutation in the Pig-a gene. Because the assay can be conducted in nontransgenic animals and the mutations accumulate with repeat dosing, we believe that the Pig-a assay could be integrated into repeat-dose toxicology studies and provides an alternative to transgenic rodent (TGR) mutation assays. The capacity and characteristics of the Pig-a assay relative to TGR mutation assays, however, are unclear. Here, using transgenic gpt delta mice, we compared the in vivo genotoxicity of single oral doses of N-ethyl-N-nitrosourea (ENU, 40 mg/kg), benzo[a]pyrene (BP, 100 and 200 mg/kg), and 4-nitroquinoline-1-oxide (4NQO, 50 mg/kg) in the Pig-a (peripheral blood) and gpt (bone marrow and liver) gene mutation assays. Pig-a assays were conducted at 2, 4, and 7 weeks after the treatment, while gpt assays were conducted on tissues collected at the 7-week terminal sacrifice. ENU increased both Pig-a and gpt mutant frequencies (MFs) at all sampling times, and BP increased MFs in both assays but the Pig-a MFs peaked at 2 weeks and then decreased. Although 4NQO increased gpt MFs in the liver, only weak, nonsignificant increases (two- or threefold above control) were detected in the bone marrow in both the Pig-a and the gpt assay. These findings suggest that further studies are needed to elucidate the kinetics of the Pig-a mutation assay in order to use it as an alternative to the TGR mutation assay. Environ. Mol. Mutagen., 2013. © 2013 Wiley Periodicals, Inc.
    Environmental and Molecular Mutagenesis 09/2013; · 3.71 Impact Factor
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    ABSTRACT: 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. 294 words.
    Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 06/2013; · 3.90 Impact Factor
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    ABSTRACT: The in vivo Pig-a mutation assay has been adapted for measuring mutation in rats, mice, monkeys, and humans. To date, the assay has been used mainly to assess the mutagenicity of chemicals that are known to be powerful point mutagens. The assay has not been used to measure the biological effects associated with ionizing radiation. In this study, we modified the Pig-a gene mutation assay (Kimoto et al. [2011b]: Mutat Res 723:36-42) and used 3-color staining with fluorescently labeled anti-CD24, anti-TER-119, and anti-CD71 to detect the Pig-a mutant frequencies in total red blood cells (RBCs) and in reticulocytes (RETs) from X-irradiated mice. Single exposures to X-irradiation resulted in dose- and time-dependent increases in Pig-a mutant frequencies, and these subsequently declined over time returning to background frequencies. The same total amount of radiation, delivered either as a single dose or as four repeat doses at weekly intervals, increased Pig-a mutant frequencies to comparable levels, reaching maxima 2-3 weeks after the single dose or 2-3 weeks after the last of the repeat doses. These increased frequencies subsequently returned to background levels. Our results indicated that the 3-color Pig-a assay was useful for evaluating the in vivo genotoxicity of radiation. Environ. Mol. Mutagen., 2012. © 2012 Wiley Periodicals, Inc.
    Environmental and Molecular Mutagenesis 08/2012; 53(8):579-88. · 3.71 Impact Factor
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    ABSTRACT: 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 Journal of Toxicological Sciences 01/2012; 37(5):943-55. · 1.38 Impact Factor
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    ABSTRACT: Recent studies indicate that the Pig-a assay is a promising tool for evaluating in vivo mutagenicity. We have developed novel rat Pig-a assays that facilitate measuring mutant frequencies in two early arising populations of blood cells, bone marrow erythroids (BMEs) and peripheral blood (PB) reticulocytes (RETs). In these assays, bone marrow cells of erythroid origin and PB red blood cells (RBCs) were identified using an antibody against rat erythroid-specific marker HIS49. In addition, RETs were selectivity enriched from PB using magnetic separation of cells positive for CD71, a transferrin receptor expressed on the surface of BMEs and RETs, but not on the surface of mature RBCs. With magnetic enrichment, more than 1 x 10(6) CD71-positive RETs could be evaluated by flow cytometry for Pig-a mutant frequency within 5 to 8 min. CD59-deficient RET and BME frequencies of more than 100 x 10(-6) and 80 x 10(-6) were detected 1 week after treating rats with 40 mg/kg N-ethyl-N-nitrosourea; by comparison, the frequency of CD59-deficient total RBCs in these rats was 13.2 x 10(-6). The frequency of spontaneous Pig-a mutant RETs and BMEs was less than 5 x 10(-6) and 15 x 10(-6), respectively. Since approximately 98% of nucleated cells in the BME fraction were erythroblasts, it should be possible to use BMEs to determine the spectrum of CD59-deficient Pig-a mutations in cells of erythroid lineage. Conducting concurrent Pig-a assays on RETs and BMEs may be useful for evaluating the in vivo mutagenicity of chemicals, especially when prolonged mutant manifestation is not feasible or when the confirmation of mutation induction is necessary.
    Environmental and Molecular Mutagenesis 12/2011; 52(9):774-83. · 3.71 Impact Factor
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    ABSTRACT: A collaborative international trial was conducted to evaluate the reproducibility and transferability of an in vivo mutation assay based on the enumeration of CD59-negative rat erythrocytes, a phenotype that is indicative of Pig-a gene mutation. Fourteen laboratories participated in this study, where anti-CD59-PE, SYTO 13 dye, and flow cytometry were used to determine the frequency of CD59-negative erythrocytes (RBC(CD59-)) and CD59-negative reticulocytes (RET(CD59-)). To provide samples with a range of mutant phenotype cell frequencies, male rats were exposed to N-ethyl-N-nitrosourea (ENU) via oral gavage for three consecutive days (Days 1-3). Each laboratory studied 0, 20, and 40 mg ENU/kg/day (n = 5 per group). Three sites also evaluated 4 mg/kg/day. At a minimum, blood samples were collected three times: predosing and on Days 15 and 30. Blood samples were processed according to a standardized sample processing and data acquisition protocol, and three endpoints were measured: %reticulocytes, frequency of RET(CD59-) , and frequency of RBC(CD59-) . The methodology was found to be reproducible, as the analysis of technical replicates resulted in experimental coefficients of variation that approached theoretical values. Good transferability was evident from the similar kinetics and magnitude of the dose-related responses that were observed among different laboratories. Concordance correlation coefficients showed a high level of agreement between the reference site and the test sites (range: 0.87-0.99). Collectively, these data demonstrate that with adequate training of personnel, flow cytometric analysis is capable of reliably enumerating mutant phenotype erythrocytes, thereby providing a robust in vivo mutation assay that is readily transferable across laboratories.
    Environmental and Molecular Mutagenesis 09/2011; 52(9):690-8. · 3.71 Impact Factor
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    ABSTRACT: Our previous rat studies indicate that the endogenous Pig-a gene is a promising reporter of in vivo mutation and potentially useful as the basis for an in vivo genotoxicity assay. The function of the Pig-a protein in the synthesis of glycosylphosphatidyl inositol (GPI) anchors is conserved in variety of eukaryotic cells, including human and rodent cells, which implies that Pig-a mutants can be measured in a similar manner in different mammalian species. In the present study, we developed a flow cytometric Pig-a assay for rapidly measuring gene mutation in the mouse. An antibody to TER-119, a specific cell-surface marker of murine erythroid lineage, was used to identify erythrocytes in peripheral blood (PB) and erythroids in bone marrow (BM). An antibody to CD24, a GPI-anchored protein, was used to identify Pig-a mutants as CD24-negative cells. CD-1 mice were administered a single dose of 100mg/kgN-ethyl-N-nitrosourea (ENU), and PB and BM were collected at 1, 2, and 4 weeks after dosing. While the Pig-a mutant frequency (MF) in PB was increased moderately at 2 and 4 weeks after ENU dosing, the Pig-a MF in BM was strongly increased starting at 1 week after the dosing, with the elevated MF persisting for at least 4 weeks after the dosing. We also used flow cytometric sorting to isolate CD24-negative erythroids from the BM of ENU-treated mice. cDNA sequencing indicated that these cells have mutations in the Pig-a gene, with base-pair substitutions typical of ENU-induced mutation spectra. The results indicate that the Pig-a mutation assay can be adapted for measuring mutation in BM erythroids and PB of mice. Taken together, the data suggest that Pig-a mutants are fixed in the BM, where they further proliferate and differentiate; erythrocytes derived from these BM Pig-a mutants transit from the BM and accumulate in PB.
    Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 07/2011; 723(1):36-42. · 3.90 Impact Factor
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    ABSTRACT: A rapid in vivo somatic cell gene mutation assay is being developed that measures mutation in the endogenous X-linked phosphatidylinositol glycan, class A gene (Pig-a). The assay detects Pig-a mutants by flow cytometric identification of cells deficient in glycosylphosphatidyl inositol (GPI) anchor synthesis. GPI-deficient, presumed Pig-a mutant cells also can be detected in a cloning assay that uses proaerolysin (ProAER) selection. Previously, we demonstrated that ProAER-resistant (ProAER(r) ) rat spleen T-cells have mutations in the Pig-a gene. In the present study, we report on a more complete analysis of ProAER(r) rat spleen T-cell mutants and describe a mutation spectrum for mutants isolated from rats 4 weeks after treatment with three consecutive doses of 35.6 mg/kg N-ethyl-N-nitrosourea (ENU). We identified a total of 55 independent mutations, with the largest percentage (69%) involving basepair substitution at A:T. The overall spectrum of Pig-a gene mutations was consistent with the types of DNA adducts formed by ENU and was very similar to what has been described for in vivo ENU-induced mutation spectra in other rodent reporter genes (e.g., in the endogenous Hprt gene and transgenic shuttle vectors). These data are consistent with the rat Pig-a assay detecting test-agent-induced mutational responses.
    Environmental and Molecular Mutagenesis 06/2011; 52(5):419-23. · 3.71 Impact Factor
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    ABSTRACT: Carbon nanoparticles, such as carbon nanotubes and fullerene (C 60), are potential candidates as leading sub-stances in nanotechnologicaîelds, but little is known about their safety. Here we examined in vivo genotoxicity of C 60 , by performing the Pig-A gene mutation assay in the peripheral blood of male C57BL/6Cr mice. Mice were given single intraperitoneal injection of 3 mg of C 60 particles in 0.5 mL suspension containing 0.1%-Tween80-saline. As a positive control for the Pig-A gene mutation assay, mice were given a single oral administration of N-nitroso-N-ethylurea. At 2 and 8 weeks after treatments, we analyzed CD24-negative and -positive red blood cells in peripheral blood and calculated Pig-A mutant frequencies. As a result, we detected no signiˆcant diŠerences in the mutant frequencies between C 60 treated and non-treated mice, in-dicating that C 60 is negative for genotoxicity in vivo in the limited target tissues assessed in this study. For the full as-sessment, we need comprehensive whole body survey on the genotoxicity of C 60 .
    Genes and Environment 03/2011; 33(1):27-31.
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    ABSTRACT: A modified flow cytometry assay for Pig-A mutant rat red blood cells (RBCs) was developed using an antibody that positively identifies rat RBCs (monoclonal antibody HIS49). The assay was used in conjunction with a flow cytometric micronucleus (MN) assay to evaluate gene mutation and clastogenicity/aneugenicity in adolescent male and female rats treated with methylphenidate hydrochloride (MPH). Sprague-Dawley rats were treated orally with 3 mg/kg MPH (70/sex) or water (40/sex) 3 x /day on postnatal days (PNDs) 29-50. Eight additional rats (4/sex) were injected i.p. with N-ethyl-N-nitrosourea (ENU) on PND 28. Blood was collected on PNDs 29, 50, and 90, and used for determining serum MPH levels and/or conducting genotoxicity assays. On the first and last days of MPH treatment (PNDs 29 and 50), serum MPH levels averaged 21 pg/microl, well within the clinical treatment range. Relative to our previously published method (Miura et al. [2008]; Environ Mol Mutagen 49: 614-629), the HIS49 Pig-A mutation assay significantly reduced the background RBC mutant frequency; in the experiments with ENU-treated rats, the modification increased the overall sensitivity of the assay 2-3 fold. Even with the increased assay sensitivity, the 21 consecutive days of MPH treatment produced no evidence of Pig-A mutation induction (measured at PND 90); in addition, MPH treatment did not increase MN frequency (measured at PND 50). These results support the consensus view that the genotoxicity of MPH in pediatric patients reported earlier (El-Zein et al. [2005]: Cancer Lett 230: 284-291) cannot be reproduced in animal models, suggesting that MPH at clinically relevant levels may be nongenotoxic in humans.
    Environmental and Molecular Mutagenesis 09/2009; 51(2):138-45. · 3.71 Impact Factor
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    ABSTRACT: We previously reported the development of an in vivo gene mutation assay using the phosphatidylinositol glycan complementation group A gene (Pig-A) as an endogenous reporter. The assay quantifies mutation in rat peripheral red blood cells (RBCs) by flow cytometric detection of cells negative for glycosylphosphatidyl inositol (GPI)-anchored protein surface markers. In this study, we examined the accumulation and persistence of Pig-A mutant RBCs in rats treated with N-ethyl-N-nitrosourea (ENU) using two dosing schedules. Male F344 rats were given single i.p. injections of 8.9, 35.6, or 142.4 mg/kg ENU or four equal weekly doses totaling 35.6 or 142.4 mg/kg ENU (8.9 mg/kgx4 or 35.6 mg/kgx4; split-dose groups). Before the treatment and through 26 weeks after the single dose or beginning the split-dose regimen, peripheral RBCs were collected and Pig-A mutant frequencies measured as RBCs negative for the GPI-anchored protein, CD59. Mean CD59-negative RBC frequencies in negative control rats ranged from 3.9 x 10(-6) to 28.7 x 10(-6) and displayed no time-related trend. With single ENU doses, CD59-negative RBC frequencies increased in a time- and dose-related manner. Maximum responses were observed beginning at 6 weeks post-treatment (57.3 x 10(-6) in the 8.9 mg/kg group; 186.9 x 10(-6) in the 35.6 mg/kg group; 759.2 x 10(-6) in the 142.4 mg/kg group), and these elevated mutant frequencies persisted to the last sampling time. In addition, splitting the dose of ENU into four weekly doses produced nearly the same mutant frequency as when given as a single dose: the maximum responses after four weekly doses of 8.9 or 35.6 mg/kg were 176.8 x 10(-6) and 683.3 x 10(-6), respectively. These results indicate that ENU-induced Pig-A mutant RBCs accumulate in a near additive fashion in rats, and once present in the peripheral blood, persist for at least 6 months. These characteristics of Pig-A mutation could be important for detecting weak mutagens by repeated or subchronic/chronic dosing protocols.
    Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 07/2009; 677(1-2):86-92. · 3.90 Impact Factor