Piga gene mutation assay

This is a laboratory-based project to identify the mutagenic capacity of oil compounds. The Hepa1c1c2 cell line is used and the endogenous Piga gene is the target DNA for mutation assessment. 5 x 10^5 Hepa1c1c7 cells were plated in 60 mm dishes and allowed to attach overnight. The following morning cells were treated with Dimethyl sulfoxide (DMSO) solvent controls (<0.1% by volume) and DMSO-Polycyclic aromatic hydrocarbon (PAH) solutions with final concentrations of 0.3 and 3.0 micromolar for 6 hours in serum-free medium. Cells were washed and expanded in culture for a minimum of 8 days for expression of the mutant phenotype. Following mutant expression 10^6 – 20^6 cells were collected and incubated with a PE-conjugated primary antibody for a GPI linked cell surface marker (CD-59). These cell populations were then immunomagnetically enriched for CD_59 negative cells by passing them through a magnetic cell separator (miltenyi LD column) following conjugation with an anti-PE paramagnetic bead. The mutant-enriched cell populations were evaluated with flow cytometry and increases in CD-59 negative cell populations were taken to represent chemically induced Piga gene mutants resulting in a CD59 phenotype. The mutagenic potency of the PAHs (Phenanthrene, Chrysene, 5,12-dimethyl-Chrysene, Dibenz[a,h]anthracene, Benzo[a]anthracene, and Benzo[k]fluoranthene) were evaluated by using Benzo[a]Pyrene as an index chemical.