The in vitro PIG-A gene mutation assay: mutagenicity testing via flow cytometry based on the glycosylphosphatidylinositol (GPI) status of TK6 cells.

The X-linked PIG-A gene is involved in the biosynthesis of the cell surface anchor GPI, and its inactivation may serve as a new marker for mutagenicity. The in vivo PIG-A gene mutation assay is currently being validated by several groups. In this study, we established a corresponding in vitro variant of the PIG-A assay applying B-lymphoblastoid TK6 cells. PE-conjugated antibodies against the GPI-anchored proteins CD55 and CD59 were used to determine the GPI status via multicolor flow cytometry. Mutant spiked TK6 cell samples were analyzed, and mutants were quantified with even small numbers being quantitatively recovered. To validate our approach, mutant spiked cell samples were analyzed by flow cytometry and proaerolysin selection in parallel, yielding a high correlation. Further, we developed a procedure to reduce the background level of preexisting mutant cells to lower than 20 in 10(6) cells to increase the sensitivity of the assay. Spontaneous rate of GPI deficiency was investigated being 0.76 × 10(-6)/cell/generation for TK6 cells. The optimal phenotype expression time after ethyl methanesulfonate treatment was found to be 10 days. We applied the in vitro PIG-A assay to demonstrate the mutagenicity of ethyl methanesulfonate, 4-nitroquinoline 1-oxide and UV-C irradiation in a dose-dependent and statistically significant manner. Pyridine and cycloheximide were included as negative controls providing negative test results up to 10 mM. These data suggest that the in vitro PIG-A assay could complement the in vivo PIG-A assay with some distinct advantages compared to other in vitro mammalian mutagenicity tests.