Evaluation of the RBC Pig-a assay and the PIGRET assay using benzo[a]pyrene in rats

Kikuzuki, Ryuta; Sato, Haruka; Fujiwara, Akira; Takahashi, Tomoko; Ogiwara, Yosuke; Sugiura, Mihoko

doi:10.1016/j.mrgentox.2016.03.010

Cited by 9 publications

(6 citation statements)

References 21 publications

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“…Furthermore, studies have measured the potential effects of complement on GPI-negative erythrocytes in rodents by measuring mutant cell frequency in immature red cells; reticulocytes, and mature erythrocytes. Although some studies have demonstrated that reticulocytes may be a better option to measure mutation post mutagenic exposure (in genotoxicology safety assessment) 41 , importantly, background levels of PIG-A mutant cells have been similar between both cell types 42 , suggesting the relatively minor role, if any, complement plays in detecting and destroying mutant cells when in such low abundance.…”

Section: Discussionmentioning

confidence: 99%

Developing a blood-based gene mutation assay as a novel biomarker for oesophageal adenocarcinoma

Haboubi

Lawrence

Rees

et al. 2019

Sci Rep

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The Phosphatidylinositol glycan class A (PIG-A) gene mutation assay phenotypically measures erythrocyte mutations, assessed here for their correlation to neoplastic progression in the gastro-oesophageal reflux disease (GORD)-Barrett’s metaplasia (BM)-oesophageal adenocarcinoma (OAC) model. Endoscopy patients underwent venipuncture and erythrocytes fluorescently stained for glycosyl phosphatidylinositol (GPI)–anchored proteins; CD55 and CD59. Using flow cytometry, GPI–anchor negative erythrocytes (mutants) were scored and compared amongst groups. The study enlisted 200 patients and 137 healthy volunteers. OAC patients had a three–fold increase in erythrocyte mutant frequency (EMF) compared to GORD patients (p < 0.001) and healthy volunteers (p < 0.001). In OAC patients, higher EMF was associated with worsening tumour staging (p = 0.014), nodal involvement (p = 0.019) and metastatic disease (p = 0.008). Chemotherapy patients demonstrated EMF’s over 19–times higher than GORD patients. Patients were further classified into groups containing those with non-neoplastic disease and those with high-grade dysplasia/cancer with 72.1% of cases correctly classified by high EMF. Within the non-neoplastic group, aspirin users had lower EMF (p = 0.001) and there was a positive correlation between body mass index (p = 0.03) and age (p < 0.001) and EMF. Smokers had EMF’s over double that of non-smokers (p = 0.011). Results suggest this test could help detect OAC and may be a useful predictor of disease progression.

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Section: Discussionmentioning

confidence: 99%

Developing a blood-based gene mutation assay as a novel biomarker for oesophageal adenocarcinoma

Haboubi

Lawrence

Rees

et al. 2019

Sci Rep

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“…Importantly, DNA sequencing results support the phenotype-genotype basis of the assay [Komoto et al, 2011; Byrne et al, 2014; Revollo et al, 2015; Krüger et al, 2016]. Much progress has also been made in regard to the number and types of chemicals investigated in the in vivo Pig-a assay, including structurally related mutagen/non-mutagen pairs [Bemis et al, 2015; Labash et al, 2016], potent clastogens [Bhalli et al, 2013a], and promutagens that must undergo metabolic activation to form DNA-reactive intermediates [Bhalli et al, 2011b and 2013b; Shi et al, 2011; Dertinger et al, 2012; Avlasevich et al, 2014; Stankowski et al, 2015; Kikuzuki et al, 2016; Koyama et al, 2016].…”

Section: Discussionmentioning

confidence: 99%

In vivo pig‐a and micronucleus study of the prototypical aneugen vinblastine sulfate

Avlasevich

Labash

Torous

et al. 2017

Environ and Mol Mutagen

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The Pig-a assay is being used in regulatory studies to evaluate the potential of agents to induce somatic cell gene mutations and an OECD test guideline is under development. A working group involved with establishing the guideline recently noted that representative aneugenic agents had not been evaluated, and to help fill this data gap Pig-a mutant phenotype and micronucleated reticulocyte frequencies were measured in an integrated study design to assess the mutagenic and cytogenetic damage responses to vinblastine sulfate exposure. Male Sprague Dawley rats were treated for twenty-eight consecutive days with vinblastine dose levels from 0.0156 to 0.125 mg/ kg/day. Micronucleated reticulocyte frequencies in peripheral blood were determined at Days 4 and 29, and mutant cell frequencies were determined at Days 24, 15, 29, and 46. Vinblastine affected reticulocyte frequencies, with reductions noted during the treatment phase and increases observed following cessation of treatment. Micronucleated reticulocyte frequencies were significantly elevated at Day 4 in the high dose group. Although a statistically significant increase in mutant reticulocyte frequencies were found for one dose group at a single time point (Day 46), it was not deemed biologically relevant because there was no analogous finding in mutant RBCs, it occurred at the lowest dose tested, and only 1 rat exceeded an upper bound tolerance interval established with historical negative control rats. Therefore, whereas micronucleus induction reflects vinblastine's well-established aneugenic effect on hematopoietic cells, the lack of a Pig-a response indicates that this tubulin-binding agent does not cause appreciable mutagenicity in this same cell type. Environ. Mol. Mutagen. 59:30-37, 2018.V C 2017 Wiley Periodicals, Inc.

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“…A single-laser FCM can be used if an alternative fluorescent label (e.g., PerCP-Cy5.5) is used on an anti-rat erythroid Ab. Refer to the RBC Pig-a assay standard protocol and an article reported by Kikuzuki et al [28] For immunomagnetic separation, cell separation magnets, such as IMagnet Cell Separation Magnet (BD Biosciences), are required. Many kinds of cell separation magnets are commercially available, but they hold up only a small number of samples at one time.…”

Section: Instrumentsmentioning

confidence: 99%

Standard protocol for the PIGRET assay, a high-throughput reticulocyte Pig-a assay with an immunomagnetic separation, used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen and Genome Society

et al. 2021

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The PIGRET assay is one of the Pig-a assays targeting reticulocytes (RETs), an in vivo genotoxicity evaluation method using flow cytometry with endogenous reporter glycosylphosphatidylinositol anchor protein. The PIGRET assay with RETs selectively enriched with anti-CD71 antibodies has several desirable features: high-throughput assay system, low background frequency of mutant cells, and early detection of mutation. To verify the potential and usefulness of the PIGRET assay for short-term testing, an interlaboratory trial involving 16 laboratories organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen and Genome Society was conducted. The collaborating laboratories assessed the mutagenicities of a total of 24 chemicals in rats using a single-treatment design and standard protocols for conducting the Pig-a assay on the total red blood cell assay and the PIGRET assay. Here the standard protocol for the PIGRET assay was described in detail.

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Evaluation of the RBC Pig-a assay and the PIGRET assay using benzo[a]pyrene in rats

Cited by 9 publications

References 21 publications

Developing a blood-based gene mutation assay as a novel biomarker for oesophageal adenocarcinoma

Developing a blood-based gene mutation assay as a novel biomarker for oesophageal adenocarcinoma

In vivo pig‐a and micronucleus study of the prototypical aneugen vinblastine sulfate

Standard protocol for the PIGRET assay, a high-throughput reticulocyte Pig-a assay with an immunomagnetic separation, used in the interlaboratory trial organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen and Genome Society

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