OXIDATIVE LUNG INJURY AND ITS PERSISTENT MITOGENOMIC EFFECTS IN HUMAN AIRWAY EPITHELIAL CELLS

The mitochondrial genome is a small, plasmid-like chromosome which encodes only 13 protein subunits yet is vital for electron transport in the mitochondrion and, therefore, vital for the existence of multicellular life. Despite this importance, mitochondrial DNA (mtDNA) is found in one of the least-protected areas of the cell, and is exposed to high concentrations of intracellular reactive oxygen species (ROS) and threat from exogenous substances and pathogens. Until recently, the quality control mechanisms that ensured the stability of the nuclear genome were thought to be minimal in the mitochondria. However, a vast network of mechanisms has been discovered that repair mtDNA lesions, replace and recycle mitochondrial chromosomes, and conduct alternate RNA processing for previously undescribed mitochondrial proteins. New mtDNA/RNA-dependent signaling pathways reveal a mostly undiscovered biochemical landscape in which the mitochondria interface with their host cells/organisms. As the myriad ways in which the function of the mitochondrial genome can affect human health have become increasingly apparent, the use of mitogenomic biomarkers (such as copy number and heteroplasmy) as toxicological endpoints has become more widely accepted. The objective of this dissertation was to investigate the role of mitochondrial genotoxicity in exacerbation and susceptibility to inflammation of the human airway epithelium. To do this, primary bronchial epithelial cells (BECs) were collected from human donors, healthy and those with chronic inflammatory lung diseases, and grown through multiple passages in serum-free culture. Disease groups were compared to healthy cells for mitogenomic markers, including expression of mtDNA genes, ultradeep sequencing for heteroplasmic frequency and mutant alleles, mtDNA lesions, and processing of polycistronic mtRNA using Nanostring probes. These results were compared to functional endpoints, including oxygen consumption rate, mitophagy, and cell viability/apoptosis, to determine whether observed mitogenomic stress had effects on epithelial cell function. The results of these studies indicated that severe chronic lung inflammation associated with fatal asthma and cystic fibrosis can cause impaired expression of mitochondrial genes, perhaps due to failed mtRNA processing. Similar endpoints were also examined following in vitro exposure of healthy BECs to nanomaterials, to examine acute genotoxic effects on mtDNA. Nanoparticle exposures had little mitogenomic effect, though carbon nanotubes did significantly decrease mtDNA expression, secondary to induction of mitophagy and decreased mitochondrial abundance.