It is proposed to investigate the hypothesis that chronic ethanol consumption results in increased oxidative damage to mtDNA and that aging may increase the susceptibility of liver mitochondria to ethanol-elicited defects in mtDNA homeostasis. Published data show that long-term ethanol feeding has a profound effect on the metabolic functioning of rat liver mitochondria. Ethanol intake results in decreased oxidative phosphorylation, structurally abnormal mitochondria and elevated levels of mitochondrially-produced reactive oxygen species. Previous studies by the investigator have established that long term exposure causes increased oxidative damage to mtDNA, as reflected by increased levels of 8-hydroxydeoxyguanosine (8-OHdG) adducts. These studies have been extended to show that 2 month old animals maintained on the Lieber-DeCarli diet for one year exhibit a 40% depletion in mtDNA content and a 3-fold increase in 8-OHdG adduct formation. In addition, increases in single strand breaks and deletions of mtDNA are also observed. Recently, a short term, chronic ethanol feeding regimen was developed where 1 year old rats are fed the Lieber-DeCarli diet for 2 months, a feeding period that is not associated with alterations in mtDNA structure in young animals. Preliminary results show that these animals exhibit a greater mtDNA depletion (greater than 60%) than that seen in young animals fed ethanol for 1 year. This suggests that aging increases the susceptibility of hepatic mitochondria to ethanol-induced alterations in mtDNA homeostasis. The proposed studies will (a) fully characterize the new feeding model with regard to mtDNA content, levels of oxidative damage and the activities of respiratory chain complexes; (b) investigate the effect of oxidative damage on the processes of mtDNA degradation and repair in old animals, in order to elucidate the biochemical mechanism(s) behind the ethanol-elicited mtDNA depletion; (c) investigate the role of ethanol and reactive oxygen species in the formation of mtDNA deletions; (d) investigate the effect of decreased mtDNA content on the production of mitochondrial transcripts and the activities of electron transport chain complexes; (e) investigate the role of mtDNA structural alterations in the formation of pathological lesions associated with alcoholic liver disease (ALD); and (f) investigate the effects of ethanol consumption on hepatic mtDNA structure in human alcoholics. Our understanding of the role of mtDNA in the pathogenesis of alcoholic liver disease will be enhanced by these in-depth analyses.
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