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

Douglas Coffin, Elizabeth Putnam, Diana Lurie, Kent Sugden

Keywords

Arsenic, DNA Damage, DNA Repair, Gene Transcription, Glutathione, Nrf2

Abstract

8-hydroxy-2'-deoxyguanosine DNA glycosylase is the first step and rate-limiting enzyme involved in the removal of 8-hydroxy-2'-deoxyguanosine via the base excision repair pathway. Transcriptional regulation of human Ogg1 is sensitive to redox changes via modulation of intracellular glutathione. In response to changes in glutathione, changes in hOgg1 transcription occur similar to genes regulated by the transcription factor Nrf2. It was determined that positions - 47 to - 44 in the hOgg1 promoter are necessary for basal transcription of Ogg1 determined by site-directed deletion. This region is capable of interacting with nuclear protein determined by binding assays. Furthermore, transcription factor Nrf2 is identified as binding to this region determined by parallel, and competition EMSA binding assays. Exposure to arsenic has also been associated with oxidative stress and damage to DNA, specifically oxo8dG. This study identified significant increases in the cellular antioxidant glutathione, and alterations in superoxide dismutase activities subsequent to arsenite exposure in actively dividing and NGF treated PC12 cells. Assessment of Ogg1 activity and mRNA levels demonstrated a significant decrease for both measures subsequent to arsenite exposure. The effect seen was due in large part to alterations in gene transcription since direct testing revealed no effect by arsenite on Ogg1 activity. Levels of oxo8dG did not significantly change subsequent to arsenite exposure, however increased trends were evident. Characterization of Sp1 binding revealed that treatment with sodium arsenite could decrease Sp1 binding at two unique Sp1 sites in the human Ogg1 promoter. In summary, transcription factor Nrf2 is an important factor in the inducible regulation of Ogg1. Transcriptional changes in Ogg1 are further dependent on the redox status of the cell. Despite the role of Nrf2 in response to oxidative stress, sodium arsenite disrupted both the transcription and activity of Ogg1 in PC12 cells. This disruption occurred despite the induction of cellular stress response via increases in GSH and Mn SOD activity. This suggests that arsenite is acting through other mechanisms potentially through disruption of the Sp1 transcription factor.

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© Copyright 2008 David Paul Cox