Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation, loss of the alveolar unit, and increased levels of oxidative damage to macromolecules, including DNA. 8-oxoguanine (8-OG) is the most common oxidative DNA lesion and its removal and repair is executed through the base excision repair pathway. The purpose of these studies was to determine whether enhancing the activity of the DNA glycosylase, OGG1, would benefit epithelial cell health by maintaining mitochondrial function following an oxidative challenge. Cigarette smoke exposure, a risk factor for COPD, enhanced oxidative DNA damage content. However, paraquat was chosen as a more specific stimulus as it intercalates within the inner mitochondrial membrane to produce excessive amounts of mitochondrial reactive oxygen species. Levels of 8-OG were measured in A549 cells using immunofluorescence and single cell phenotypic analysis was undertaken by a high content imaging platform. Transduction of A549 cells with full length-OGG1 baculovirus lowered the maximal levels of 8-OG in mtDNA compared to null virus control cells. Conversely, administration of OGG1 siRNA rendered the cells more vulnerable to paraquat, increasing 8-OG content. Exemplars of small molecule OGG1 activators identified through a high-throughput-screen were shown to reduce paraquat-induced 8-OG formation. Moreover, OGG1 activators improved paraquat-induced loss of mitochondrial membrane potential, while paraquat-induced cytochrome c translocation to the nucleus was blocked. The paraquat-stimulated decline in the cellular energy state, i.e., the ATP/ADP ratio, was prevented in the presence of the OGG1 activators. Paraquat-induced changes in mitochondrial dynamics were blocked with the OGG1 activators, but did not affect mitochondrial mass. These data provide evidence of cytoprotection from oxidant-induced mtDNA damage when OGG1 protein is increased or activated, suggesting that the base excision repair pathway may be a target for potential small molecule intervention in COPD.
|Date of Award||1 Oct 2016|
- University Of Strathclyde
|Sponsors||University of Strathclyde|
|Supervisor||Susan Pyne (Supervisor) & Ben Pickard (Supervisor)|