Year of Award

2008

Document Type

Dissertation

Degree Type

Doctor of Philosophy (PhD)

Degree Name

Toxicology

Department or School/College

Department of Biomedical and Pharmaceutical Sciences

Committee Chair

Fernando Cardozo-Pelaez

Commitee Members

Diana Lurie, Richard Bridges, Darrell Jackson, Kent Sugden

Keywords

DNA damage, free radicals, GTP, guanosine, guanylyl cyclase, oxidative stress

Abstract

Oxidation of the guanosine (G) moiety, yielding the oxidized lesion 8-hydroxy-2'-deoxyguanosine (oxo82dG), in DNA has become a hallmark biomarker in assessing cellular outcomes induced by oxidative stress. It is well established that the guanosine nucleotide triphosphate pool is also susceptible to oxidative stress and suggested to be more available for oxidation than DNA due to the lack of protective histones and robust repair mechanisms for reducing the levels of all the products of oxidation. The oxidation of guanosine in the nucleotide triphosphate pool, resulting in oxidized guanosine 5'-triphoshate (oxo8GTP), has been overlooked due to the lack of a reliable method. Oxo8GTP has been shown to precede oxidation to G incorporated into DNA and modulate cell processes such as G-protein signaling and RNA synthesis. Evidence is presented in this study of a reliable method to quantify oxo8GTP, a proposed mechanism for the oxidative modification of GTP in the presence of copper and L-ascorbic acid, and evidence of oxo8GTP as an inhibitor of soluble guanylyl cyclase (sGC). A significant induction of oxo8GTP in cell-free preparations as well as in PC12 and HEK 293T cells exposed to physiologically relevant oxidative conditions generated with 10 ?M copper sulphate and 1mM L-Ascorbic Acid (Cu/Asc) is also reported. Exposure to oxidative conditions by Cu/As leads to elevations in oxo8GTP significant enough to result in a reduction of the sGC product, cyclic guanosine monophosphate (cGMP), by as much as half in pure sGC and PC12 cells. GTP is protected from oxidation in the presence of reduced glutathione and this subsequently rescues sGC activity. This suggests that oxo8GTP is produced by free radicals in vivo and can significantly impact neuronal cell functions regulated by sGC activity in the central nervous system such as synaptic plasticity. Alterations in copper homeostasis and oxidative stress have been implicated in several neurodegenerative disorders including Alzheimer's and Parkinson's diseases as well as Amyotrophic Lateral Sclerosis. Based upon evaluation of the data presented herein, we hypothesize that neuronal deficiencies in such disorders might be due to oxidation of the GTP pool and the ensuing effects on neuronal function.

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© Copyright 2008 Celeste Maree Bolin