The Pig‐a Gene Mutation Assay in Mice and Human Cells: A Review

Abstract

This MiniReview describes the principle of mutation assays based on the endogenous Pig-a gene and summarizes results for two species of toxicological interest, mice and human beings. The work summarized here largely avoids rat-based studies, as are summarized elsewhere. The Pig-a gene mutation assay has emerged as a valuable tool for quantifying in vivo and in vitro mutational events. The Pig-a locus is located at the X-chromosome, giving the advantage that one inactivated allele can give rise to a mutated phenotype, detectable by multicolour flow cytometry. For in vivo studies, only minute blood volumes are required, making it easily incorporated into ongoing studies or experiments with limited biological materials. Low blood volumes also allow individuals to serve as their own controls, providing temporal information of the mutagenic process, and/or outcome of intervention. These characteristics make it a promising exposure marker. To date, the Pig-a gene mutation assay has been most commonly performed in rats, while reports regarding its usefulness in other species are accumulating. Besides its applicability to in vivo studies, it holds promise for genotoxicity testing using cultured cells, as shown in recent studies. In addition to safety assessment roles, it is becoming a valuable tool in basic research to identify mutagenic effects of different interventions or to understand implications of various gene defects by investigating modified mouse models or cell systems. Human blood-based assays are also being developed that may be able to identify genotoxic environmental exposures, treatment- and lifestyle-related factors or endogenous host factors that contribute to mutagenesis.

Mode of action-based risk assessment of genotoxic carcinogens

“…Despite the fact that mutations that lead to a loss of GPI anchors have not been well characterized, it is a gene mutation that constitutes the genetic endpoint addressed so that this test could be a follow-up test to investigate positive Ames test results in vivo. Whereas this assay can be used in various animal species and cell types in vitro and in vivo (Olsen et al 2017), it is currently being mostly used to quantify CD59-deficient (presumed Pig-a mutant) erythrocytes in the peripheral blood of rats. Thus, it lends itself to being integrated into repeated-dose toxicity studies, since only a minimal amount of peripheral blood needs to be collected for analysis so that even a longitudinal analysis is feasible (Godin-Ethier et al 2015).…”

“…Nevertheless, this would require very sensitive in vivo mutagenicity test systems, which are currently not available. One promising approach could be the newly established PIG-A test, which could even be implemented in repeated-dose toxicity studies, since only sampling of a small amount of blood is required for this test (for a recent review see Olsen et al 2017).…”

Direct Quantification of in vivo Mutagenesis and Carcinogenesis Using Duplex Sequencing

“…TGR assays are complex to establish, require special breeding programs, and any spectral analysis requires manually picking and sequencing hundreds of plaques 15 . The only in vivo mutagenesis assay that does not depend on in vitro selection, the Pig-a Gene Mutation Assay, is restricted to one tissue type, requires bioavailability to the bone marrow compartment, cannot be used for spectrum analysis, and necessitates access to flow cytometery equipment 43 . In contrast, next generation DNA sequencing platforms are widely available and can be readily automated to handle thousands of samples per day.…”

Genotoxic effects of high dose rate X‐ray and low dose rate gamma radiation in ApcMin/+ mice

“…Furthermore, mutagenicity is one of several hallmarks involved in cancer development (Hanahan and Weinberg, ; Hanahan and Weinberg, ). The methods used to investigate these endpoints are either established as OECD Technical Guidance (TG) protocols (OECD TG 474, MN assay and TG 489, SCGE assay) or under validation to become such protocols [ Pig‐a gene mutation assay, reviewed in (Olsen et al, )]. The three assays measure distinct but complementary facets of genotoxicity; the initial DNA damage levels with potential to become DNA mutations and the mutation analyses covering two classes of mutation.…”