Manifestation of Pig-a mutant bone marrow erythroids and peripheral blood erythrocytes in mice treated with N-ethyl-N-nitrosourea: Direct sequencing of Pig-a cDNA from bone marrow cells negative for GPI-anchored protein expression

Kimoto, Tsunenobu; Suzuki, Kumiko; Kobayashi, Xiao mei; Dobrovolsky, Vasily N.; Heflich, Robert H.; Miura, Daisuke; Kasahara, Yoshinori

doi:10.1016/j.mrgentox.2011.03.016

Cited by 58 publications

(53 citation statements)

References 18 publications

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“…Much less is known about the types of mutations detected by the Pig-a gene. To date, sequence analysis of Pig-a mutants from rodent assays [Miura et al, 2008Kimoto et al, 2011] and analysis of PIG-A mutations associated with the human acquired disease paroxysmal nocturnal hemoglobinuria [Nafa et al, 1998;Nishimura et al, 1999;Mortazavi et al, 2003] have mainly detected base pair substitutions.…”

Section: Discussionmentioning

confidence: 99%

Report on stage III Pig‐a mutation assays using benzo[a]pyrene

Bhalli

Shaddock

Pearce

et al. 2011

Environ and Mol Mutagen

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Genotoxicity assays were conducted on rats treated with benzo[a]pyrene (BaP) as part of Stage III of a validation study on the Pig-a gene mutation assay. Assays were performed at the U.S. FDA-NCTR and Bayer-Germany. Starting on Day 1, groups of five 6- to 7-week-old male Fischer 344 (F344, used at FDA-NCTR) and Han Wistar rats (Bayer) were given 28 daily doses of 0, 37.5, 75, or 150 mg/kg BaP; blood was sampled on Days -1, 4, 15, 29, and 56. Pig-a mutant frequencies were determined on Days -1, 15, 29, and 56 in total red blood cells (RBCs) and reticulocytes (RETs) as RBC(CD59-) and RET(CD59-) frequencies; percent micronucleated-RETs (%MN-RET) were measured on Days 4 and 29. RBC(CD59-) and RET(CD59-) frequencies increased in a dose- and time-dependent manner, producing significant increases by Day 29 in both rat models. The responses for RETs were stronger than those for RBCs, and the responses in F344 rats were stronger than in Han Wistar rats. BaP also produced significant increases in %MN-RET frequency at Days 4 and 29, with the responses being greater in F344 than Han Wistar rats. The overall findings were consistent with those of the reference laboratory using Han Wistar rats. Finally, mutation assays performed on splenocytes from Day 56 F344 rats indicated that BaP mutant frequencies were three to fivefold higher for the Hprt gene than the Pig-a gene. The results indicate that the Pig-a RET and RBC assays are reproducible, transferable, and show promise for integrating gene mutation into 28-day repeat-dose studies.

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Section: Discussionmentioning

confidence: 99%

Report on stage III Pig‐a mutation assays using benzo[a]pyrene

Bhalli

Shaddock

Pearce

et al. 2011

Environ and Mol Mutagen

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“…In a previous study, we developed our Pig-a assay based on Kimoto's Pig-a gene-mutation assay (Kimoto et al, 2011 using mouse red blood cells (RBCs) stained with 3 markers, including a fluorescently labeled anti-CD24 antibody, an anti-TER-119 antibody, and an anti-CD71 antibody (Ohtani et al, 2012). It has become a useful method for detecting the in vivo genotoxic effects of ionizing radiation on impaired hematopoietic stem cells.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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-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 radiationinduced 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.

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“…During our efforts to develop a flow cytometric Pig-a assay for measuring gene mutation in N-ethyl-N-nitrosourea (ENU) treated rodents, we demonstrated increased frequencies of GPI-anchored-proteindeficient peripheral RBCs, bone marrow erythroids, and splenocytes (Kimoto et al, 2011a(Kimoto et al, , 2011bMiura et al, 2008aMiura et al, , 2008b. The use of non-nucleated RBCs to measure Pig-a gene mutation has been supported by sequence analysis of the Pig-a gene in GPI-anchored-protein-deficient bone marrow erythroids, which are the presumed precursors of PB RBCs (Kimoto et al, 2011b). Related to this, we have proposed a model for Pig-a mutant manifestation to account for the accumulation and persistence of GPI-deficient Pig-a mutant RBCs observed in ENU treated rats .…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2012

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-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.

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Manifestation of Pig-a mutant bone marrow erythroids and peripheral blood erythrocytes in mice treated with N-ethyl-N-nitrosourea: Direct sequencing of Pig-a cDNA from bone marrow cells negative for GPI-anchored protein expression

Cited by 58 publications

References 18 publications

Report on stage III Pig‐a mutation assays using benzo[a]pyrene

Report on stage III Pig‐a mutation assays using benzo[a]pyrene

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

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

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