Using a precision charged particle microbeam, the applicant and his co-investigators have shown that the frequencies of induced mutations and chromosomal changes in populations where some known fractions of nuclei were hit are consistent with non-hit cells contributing significantly to the response. In fact, irradiation of 10% of a mammalian cell population with a single alpha particle per cell results in a mutant yield similar to that observed when all of the cells in the population are irradiated. While gap junctional communication has been shown to play an important role in the bystander response, the precise mechanism is not known. Using cDNA microarrays, the applicant has shown that the COX-2 enzyme is consistently elevated in bystander cells. Furthermore, preliminary evidence suggests that reactive nitrogen species may be involved in the signaling process. This raised the following questions: Are peroxynitrite anions involved in the bystander effect? Does this radical generating process involve mitochondrial damage? How does an increase in COX-2 enzymes relate to the bystander process? Can cytoplasmic irradiation induce bystander mutagenic effect in mammalian cells in a manner similar to what the applicant has recently demonstrated with nuclear traversal of normal human bronchial epithelial (NHBE) cells? And finally, can bystander signal induce genomic instability in mammalian cells? To address these issues, a series of 8 inter-related specific aims are proposed to address the 5 testable hypotheses. Mutations will be scored at the CD59 locus of the AL cells and G2PCC will be scored in NHBE cells. The proposed studies will help to address the mechanism of bystander mutagenesis in mammalian cells. Together with the cytoplasmic genotoxicity study, this project will address, in the context of this program project, some of the fundamental issues regarding both the target and radiation dose effect and are likely to have a significant impact on our current understanding of radiation risk assessment.
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