Archives of Toxicology

, Volume 92, Issue 4, pp 1609–1623 | Cite as

The development of an in vitro Pig-a assay in L5178Y cells

  • Rhiannon David
  • Emily Talbot
  • Bethany Allen
  • Amy Wilson
  • Usman Arshad
  • Ann Doherty
Genotoxicity and Carcinogenicity

Abstract

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.

Keywords

Mutation GPI anchor Flow cytometry Micronucleus Sequencing 

Notes

Compliance with ethical standards

Conflict of interest

The authors disclose no potential conflicts of interest.

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Rhiannon David
    • 1
  • Emily Talbot
    • 1
  • Bethany Allen
    • 1
  • Amy Wilson
    • 1
  • Usman Arshad
    • 1
  • Ann Doherty
    • 1
  1. 1.Genetic Toxicology, Discovery Safety, Drug Safety and Metabolism, IMED Biotech UnitAstraZenecaCambridgeUK

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