Characterization of the flow cytometry mutation assay and its use in novel genotoxicity studies
Date
2009
Journal Title
Journal ISSN
Volume Title
Abstract
The flow cytometry mutation assay (FCMA) has been previously demonstrated to be a rapid and sensitive assay for measuring mutations induced by a wide variety of genotoxic agents. After treatment with a mutagen, the mutant fraction measured by the FCMA increased to a peak over time and subsequently decreased to a stable plateau. Using ethyl methanesulfonate (EMS), ionizing radiation (IR) and asbestos, I determined that the return to normal cell survival is an indicator of peak mutant expression. Decreased survival significantly contributes to the decline in the mutant fraction and confirms that mutations that are not clonogenically viable are measured by the FCMA on the peak day of expression. Also, I analyzed clones isolated from several CD59- regions and generated mutant spectra for EMS using flow cytometry. I then investigated hypoxia induced mutagenesis. It has been previously shown that hypoxic stress can generate DNA damage and mutations which are likely caused by reactive oxygen species (ROS). Here I demonstrate that the oxygen radical scavenger dimethyl sulfoxide significantly decreased cell killing and mutagenesis after hypoxia treatment, supporting the concept that ROS are responsible for hypoxia induced mutations. I also investigated the effects of silencing of DNA repair proteins on cell survival, cell cycle and mutagenesis. The knockdown of homologous recombination repair protein Rad51C slightly increased sensitivity to IR and drastically increased killing by EMS treatment. Rad51C knockdown also caused a significant G2 phase buildup after EMS treatment. Silencing of the non-homologous end joining (NHEJ) protein Ku80 increased cell sensitivity to IR and decreased the mutant yield after EMS treatment. This implies that EMS generates significant double strand breaks (DSBs) during S phase that are possibly repaired by NHEJ. In summary, I have demonstrated that the FCMA is a fast and reliable method to measure mutagenesis induced by an agent and to quantify the degree of damage by obtaining a mutant spectrum. I have used this assay to investigate mechanisms of mutagenesis by EMS, IR, asbestos and hypoxia and evaluated the effects of DNA repair pathways on cell cycle, cell killing and mutant induction.
Description
Rights Access
Subject
flow cytometry
genotoxicity
mutation assays
radiation
reactive oxygen species
molecular biology
cellular biology