The development of an in vitro Pig-a assay in L5178Y cells
| Autor: | Usman Arshad, Amy Wilson, Bethany Allen, Ann T. Doherty, Rhiannon David, Emily Talbot |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Ethyl methanesulfonate Health Toxicology and Mutagenesis Population Cycloheximide Toxicology Flow cytometry 03 medical and health sciences chemistry.chemical_compound Mice In vivo medicine Tumor Cells Cultured Animals Mutation frequency education education.field_of_study biology medicine.diagnostic_test Mutagenicity Tests Membrane Proteins General Medicine Molecular biology In vitro 030104 developmental biology chemistry biology.protein Antibody Mutagens |
| Zdroj: | Archives of toxicology. 92(4) |
| ISSN: | 1432-0738 |
| Popis: | A recent flow cytometry-based in vivo mutagenicity assay involves the hemizygous phosphatidylinositol class A (Pig-a) gene. Pig-a forms the catalytic subunit of N-acetylglucosaminyltransferase required for glycophosphatidylinositol (GPI) anchor biosynthesis. Mutations in Pig-a prevent GPI-anchor synthesis resulting in loss of cell-surface GPI-linked proteins. The aim of the current study was to develop and validate an in vitro Pig-a assay in L5178Y mouse lymphoma cells. Ethyl methanesulfonate (EMS)-treated cells (186.24-558.72 µg/ml; 24 h) were used for method development and antibodies against GPI-linked CD90.2 and stably expressed CD45 were used to determine GPI-status by flow cytometry. Antibody concentration and incubation times were optimised (0.18 µg/ml, 30 min, 4 °C) and Zombie Violet™ (viability marker; 0.5%, 30 min, RT) was included. The optimum phenotypic expression period was 8 days. The low background mutation frequency of GPI-deficiency [GPI(-)] in L5178Y cells (0.1%) constitutes a rare event, thus flow cytometry acquisition parameters were optimised; 104 cells were measured at medium flow rate to ensure a CV ≤ 30%. Spiking known numbers of GPI(-) cells into a wild-type population gave high correlation between measured and spiked numbers (R2 0.999). We applied the in vitro Pig-a assay to a selection of well-validated genotoxic and non-genotoxic compounds. EMS, N-ethyl-N-nitrosourea and 4-nitroquinoline-N-oxide dose dependently increased numbers of GPI(-) cells, while etoposide, mitomycin C, and a bacterial-specific mutagen did not. Cycloheximide and sodium chloride were negative. Sanger sequencing revealed Pig-a mutations in the GPI(-) clones. In conclusion, this in vitro Pig-a assay could complement the in vivo version, and follow up weak Ames positives and late-stage human metabolites or impurities. |
| Databáze: | OpenAIRE |
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