The in vivo pig‐a gene mutation assay, a potential tool for regulatory safety assessment

Abstract

The Pig-a (phosphatidylinositol glycan, Class A) gene codes for a catalytic subunit of the N-acetylglucosamine transferase complex involved in an early step of glycosylphosphatidyl inositol (GPI) cell surface anchor synthesis. Pig-a is the only gene involved in GPI anchor synthesis that is on the X-chromosome, and research into the origins of an acquired genetic disease involving GPI anchor deficiency (paroxysmal nocturnal hemoglobinuria) indicates that cells lacking GPI anchors, or GPI-anchored cell surface proteins, almost always have mutations in the Pig-a gene. These properties of the Pig-a gene and the GPI anchor system have been exploited in a series of assays for measuring in vivo gene mutation in blood cells from humans, rats, mice, and monkeys. In rats, flow cytometric measurement of Pig-a mutation in red blood cells requires microliter volumes of blood and data can be generated in hours. Spontaneous mutant frequencies are relatively low (<5 × 10(-6)) and rats treated with multiple doses of the potent mutagen, N-ethyl-N-nitrosourea, display Pig-a mutant frequencies that are close to the sum of the frequencies produced by the individual exposures. A general observation is that induced mutant frequencies are manifested earlier in reticulocytes (about 2 weeks after treatment) than in total red blood cells (about 2 months after exposure). Based on data from a limited number of test agents, the assay shows promise for regulatory applications, including integration of gene mutation measurement into repeat-dose toxicology studies.

Testing the Mutagenicity Potential of Chemicals

“…An in vivo assay with potential for regulatory safety assessment is the Pig-A gene mutation assay [52]. This assay is capable of measuring in vivo gene mutations at the Pig-A gene, which cause failure of 'GPI anchors', resulting in the absence of specific surface markers on the exterior of peripheral blood cells.…”

Scientific opinion on genotoxicity testing strategies applicable to food and feed safety assessment

“…Although this assay can be carried out on a number of species and cell types at present only blood cells have been successfully used. Most published studies have used rat red blood cells and reticulocytes (reviewed in Dobrovolsky et al, 2010). The test protocol requires small blood volumes (µlitres) if a flow cytometric assay is carried out and this makes integration with, for instance, repeat-dose toxicology tests highly feasible.…”

The effects of methyl-donor deficiency on mutation induction and transgenerational instability in mice

“…A perfect screening test would be able to utilize high throughput methodology and would be able to detect mutations in a number of animal species, including humans. Currently the most frequently used mutation assays in vivo are the numerous bacterialtransgene rodent models such as the lacI or lacZ, the lymphocyte hypoxanthine-guanine phosphoribosyltransferase (Hprt) assay, and the human Glycophorin A (GPA) assay (Miura et al, 2008;Dobrovolsky 2010b).…”