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Thesis Details
TitleOxidative damage to mitochondria on ageing in rats
AuthorDavies, Stefan M. K.
InstitutionUniversity of Otago
Date2007
AbstractAgeing is a complex phenomenon, characterised by progressive loss of function, decreasing resistance to age-associated pathologies and stress, and increasing rates of mortality. The Free Radical Theory of Ageing implicates reactive oxygen and nitrogen species (ROS/RNS) generation as being integral to the ageing process, subjecting the organism to oxidative stress. Oxidative damage to biomolecules is suggested to be causative in the formation of ageing-associated phenotypes, dysregulation and dysfunction. Mitochondria are responsible for the production of the majority of ROS/RNS through normal functioning of the respiratory chain. Previous studies have reported increasing mitochondrial dysfunction with age, including oxidative damage to protein, lipid and DNA. Thus, mitochondrial dysfunction is considered by many to be central to the Free Radical Theory of Ageing. However, there are conflicting data on changes in mitochondrial and cellular function and damage on ageing. To investigate the role of mitochondrial protein oxidative damage in ageing, the heart, brain and liver of young (~ 2 month-old) and old (24 month-old) Wistar rats were fractionated into homogenate, cytosolic and mitochondrial fractions. Mitochondrial function was evaluated by measuring activity of the oxidative phosphorylation Complexes I-V, and correlating activity with quantitation of Complex subunits. The activities of the electron transport chain Complexes (I-IV) were largely unchanged on ageing, and no significant differences were seen in the protein levels of nuclear-encoded Complex I-IV subunits. There was a ~ 40% decrease in ATP synthase activity in heart and liver mitochondria from old rats as compared to young, but no change in the level of the Complex V nuclear-encoded subunit. These results suggest the decreased activity is due to modification of Complex V in heart and liver mitochondria on ageing, rather than changes in expression. Oxidative stress is a common cause of mitochondrial dysfunction, and is often accompanied by an increase in cellular antioxidant defences. Expression of the mitochondrial antioxidant enzyme, MnSOD, was found to be increased in the liver (+ 74%) and heart (+ 82%), but not brain, in old rats, suggesting oxidative stress in these organs on ageing in rats. To investigate generalised protein oxidative damage accumulation on ageing, whole tissue homogenate, cytosol and mitochondria were isolated from young and old heart, brain and liver. These fractions were assayed for three markers of protein oxidative damage: protein carbonyl content (a marker for generalised oxidative damage occurring via attack by many ROS/RNS), and ortho-tyrosine and meta-tyrosine accumulation (two markers specific for hydroxyl radical attack on phenylalanine). There were no consistent age-related changes in these biomarkers in any tissues, and no consistent significant differences between cytosolic and mitochondrial protein oxidative damage for any of the three tissues in the two age cohorts. Mitochondria were further subfractionated into membrane-enriched and matrix-enriched subfractions, but again, protein oxidative damage markers were largely unchanged on ageing. These results suggest that there is no common pattern of mitochondrial dysfunction during ageing in rats. Increased mitochondrial oxidative stress is a feature of ageing, but generalised protein oxidative damage is neither necessary nor sufficient for development of the ageing phenotype.
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