Document Type

Dissertation

Date of Degree

2013

Degree Name

PhD (Doctor of Philosophy)

Degree In

Human Toxicology

First Advisor

Douglas Spitz

Abstract

Exposure of mammalian cells to ionizing radiation is believed to affect mitochondrial metabolism, which can lead to increased levels of superoxide (O2*-). Sirtuin3 (Sirt3) is a key mitochondrial protein deacetylase thought to play a significant role in the maintenance of mitochondrial integrity and oxidative metabolism in response to radiation. Here I show evidence that Sirtuin3 regulates mitochondrial oxidative metabolism in unirradiated mouse embryonic fibroblasts (MEFs) as well as regulates survival and steady-state levels of O2*- following exposure to radiation. This work also identifies novel, small molecule, superoxide dismutase mimetics as being capable of ameliorating the alterations in mitochondrial oxidative metabolism and cell toxicity following exposure to radiation. When compared to WT MEFs, unirradiated Sirt3-/- MEFs demonstrate increased levels of reactive oxygen species (ROS), hyperpolarized mitochondrial membranes, increased oxygen consumption, higher glycolytic flux and lower ATP levels demonstrating that the loss of Sirt3 profoundly regulates mitochondrial metabolism. Furthermore, increased levels of mitochondrial fragmentation were also observed in irradiated Sirt3-/- MEFs, relative to un-irradiated controls. When exposed to 2 Gy of X-rays, Sirt3-/- MEFs also demonstrated increased steady-state levels of O2*- as indicated by DHE oxidation using flow cytometry and confocal microscopy. Sirt3-/- MEFs irradiated with 1-2 Gy also showed significantly increased clonogenic cell killing as well as a compromised ability to induce an adaptive response following exposure to a 0.1 Gy priming dose, as compared to Sirt3 WT MEFs. Increased levels of O2*- seen following irradiation of Sirt3-/- MEFs were ameliorated by the use of two superoxide dismutase mimetics (GC4401 and GC4403) but not the catalytically inactive form of the compound (GC4404) suggesting the causal role of the metabolic production of O2*- in radiation-induced toxicity. In addition, alterations in the activity of Complex II of the electron transport chain were also observed in the Sirt3-/- MEFs suggesting that Sirt3 may be essential in the maintenance of mitochondrial electron transport chain (ETC) activity following exposure to radiation. Collectively, this work demonstrates that Sirt3 plays a key role in regulating the levels of mitochondrial O2*- that may affect the overall maintenance of mitochondrial oxidative metabolism and survival following exposure to ionizing radiation. (Supported by DOE DE-SC0000830).

Pages

xi, 144 pages

Bibliography

Includes bibliographical references (pages 130-143).

Copyright

Copyright 2013 Kranti Ashok Mapuskar

Included in

Toxicology Commons

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