Impaired adaptive resynthesis and prolonged depletion of hepatic mitochondrial DNA after repeated alcohol binges in mice

Background & Aims: A single dose of alcohol causes transient hepatic mitochondrial DNA (mtDNA) depletion in mice followed by increased mtDNA synthesis and an overshoot of mtDNA levels. We determined the effect of repeated alcohol binges on hepatic mtDNA in mice. Methods: Ethanol (5 g/kg) was administered by gastric intubation daily for 4 days, and mtDNA levels, synthesis, and integrity were assessed by slot blot hybridization, in organello [H-3]deoxythymidine triphosphate incorporation, and long polymerase chain reaction analysis, respectively. Results: mtDNA levels were decreased for 48 hours after the last dose, with no overshoot phenomenon later on. Two and 24 hours after the fourth dose, long polymerase chain reaction experiments showed DNA lesions that blocked the progress of the polymerases and in organello mtDNA synthesis was decreased, although DNA polymerase gamma activity was unchanged with synthetic templates. Mitochondria exhibited ultrastructural abnormalities, and respiration was impaired 2 and 24 hours after the fourth binge. Cytochrome P450 2E1, mitochondrial generation of peroxides, thiobarbituric acid reactants, and ethane exhalation were increased. Conclusions: After repeated doses of ethanol, the accumulation of unrepaired mtDNA lesions (possibly involving lipid peroxidation-induced adducts) blocks the progress of polymerase gamma on mtDNA and prevents adaptive mtDNA resynthesis, causing prolonged hepatic mtDNA depletion.