We propose to study the chemical spectrum and biological consequences of deoxyribose oxidation produced by ionizing radiation and chemical oxidants in isolated DNA and in cells. DNA oxidation is strongly associated with the pathophysiology of cancer and aging, with the bulk of research in this area focused on nucleobase lesions. However, there is growing evidence that deoxyribose oxidation plays a critical role in the genetic toxicology of oxidative stress, including involvement in complex DNA lesions, cross-linking with DNA repair proteins and the formation of endogenous DNA adducts. In spite of this evidence, there have been few studies that address the chemistry of deoxyribose oxidation in cells. We propose to develop sensitive analytical techniques to quantify the spectrum of deoxyribose oxidation products in isolated DNA and in cells exposed to different oxidizing agents, and to define the cellular response to these products.
Aim #1 : Development of analytical techniques to quantify deoxyribose oxidation products. With abundant support from preliminary studies, we will develop GC/MS strategies for quantifying oxidation products arising from the 1'-, 3'-, 4'-, and 5'-positions of deoxyribose.
Aim #2 : Development of alpha-particle sources. A major goal of our studies is to compare and contrast the chemistry of deoxyribose oxidation caused by alpha- and gamma-radiation. Limited access to alpha-radiation facilities has motivated us to develop versatile 241Am alpha-particle sources for irradiation of isolated DNA and cells.
Aim #3 : Characterization of deoxyribose oxidation products in purified DNA. The methods and radiation sources developed in Aims #1 and #2 will be applied to quantifying deoxyribose oxidation products in isolated DNA. The goals here are: (1) to lay the groundwork for studies in cells; and (2) to test hypotheses related to the chemical mechanisms of deoxyribose oxidation by ionizing radiation and chemical oxidants.
Aim #4 : Defining the chemistry and biology of deoxyribose oxidation in cells. Observations made in isolated DNA will now be carded into cells. There are two objectives here: (1) to define the effect of the cellular environment on the chemistry of deoxyribose oxidation; and (2) to define the role of deoxyribose oxidation in the cellular responses to oxidative stress.
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