In Depth
In Vivo MutaFlow Kits

The Pig-a Mutation Assay allows investigators to measure mutant cell frequencies in peripheral blood samples. As with the Hprt reporter gene, Pig-a is an endogenous gene located on the X-chromosome. It is one of several genes involved in glycosyl-phosphatidylinisotol (GPI) anchor synthesis. Mutation of any one of these genes may cause GPI anchor deficiency.

It is important to note however, that all the other genes involved in GPI anchor synthesis are autosomal. In this case, mutations in both alleles would have to occur to prevent expression of GPI anchors, which is very unlikely. Therefore, the inability to anchor GPI-linked proteins to the cell membrane serves as a reliable reporter of Pig-a mutation.

To our knowledge, Luzzatto and colleagues were the first to propose that the Pig-a gene (in their case, the human PIG-A gene) might serve as a reporter of in vivo mutation. More recently, scientists at Litron, FDA-NCTR, and Teijin Pharma have described the development of flow cytometry-based rodent assays using Pig-a as a mutation reporter [Bryce et al., 2008; Miura et al., 2008a,b; Phonethepswath et al., 2008]. This work demonstrated an increase in erythrocytes expressing the Pig-a mutant phenotype for rats and mice treated with prototypical mutagens.

The following observations, from these early experiments, have contributed to the current enthusiasm for Pig-a-based mutation assays:

• Compatible with standard rodent strains.
• The incorporation of a nucleic acid dye allows the percentage of mutant phenotype cells to be measured in both newly formed reticulocytes (RETs) and the total erythrocyte pool.
• Mutant frequencies measured over time indicate that mutant phenotype erythrocytes are not actively removed from the bloodstream, at least in rodent models. This is unlike Hprt lymphocyte mutants or micronucleated rat erythrocytes.
• Pig-a mutants accumulate with repeat dosing, making the assay compatible with many experimental designs, including pivotal 28-day toxicology studies [Miura et al., 2009; Dertinger et al., 2010].

The majority of work to date has focused on rat and mouse models. The cell type of choice has been peripheral blood erythrocytes and reticulocytes, owing to the ease by which sufficient numbers of cells can be collected from on-going studies. The potential to turn this into a cross-species endpoint, including humans, appears very high.

An international multi-laboratory trial, led by Litron, is focused on testing the transferability, reproducibility, and relative sensitivity of the rat Pig-a mutation assay. After enrollment (Stage I), participants were asked to conduct an acute (3 dose) treatment with N-ethyl-N-nitrosourea (ENU) and monitor Pig-a mutant frequencies in RETs and total RBCs at designated sampling times. The results of these Stage II experiments are provided in a paper by Dertinger et al. [2011b].

Following up on its potential for integration into subchronic toxicology assays, the Stage III trial utilized a 28-day repeat-dosing schedule and evaluated the ability of the assay to detect five standard mutagens: benzo[a]pyrene (BaP) [Bhalli et al., 2011b]; ENU [Cammerer et al., 2011]; N-methyl-N-nitrosourea [Lynch et al., 2011]; 4-nitroquinoline-1-oxide [Stankowski et al., 2011]; and 7,12-dimethylbenz[a]anthracene [Shi et al., 2011]. Stage IV work is now underway, and these experiments are intended to test a wider range of agents, including those expected to be weak or negative, using both 28-day and 3-day dosing protocols.

The papers contained in a Special Issue of Environmental & Molecular Mutagenesis (vol. 52, no. 9, 2011) are representative of the range of activities associated with this validation effort. A paper by Schuler and colleagues [2011] describes the ILSI-HESI (International Life Sciences Institute-Health and Environmental Sciences Institute) perspective on the use of a Pig-a mutation assay for regulatory evaluations. A major portion of the special issue is devoted to reports of Stage II, III, and IV studies from this trial.