FIALURIDINE AND ITS METABOLITES INHIBIT DNA-POLYMERASE-GAMMA AT SITESOF MULTIPLE ADJACENT ANALOG INCORPORATION, DECREASE MTDNA ABUNDANCE, AND CAUSE MITOCHONDRIAL STRUCTURAL DEFECTS IN CULTURED HEPATOBLASTS

The thymidine analog fialuridine oxy-2-fluoro-beta-D-arabinofuranosyl) -5-iodouracil (FIAU)] was toxic in trials for chronic hepatitis B infe ction, One mechanism postulated that defective mtDNA replication was m ediated through inhibition of DNA polymerase-gamma (DNA pol-gamma) by FIAU triphosphate (FIAUTP) or by triphosphates of FIAU metabolites, In hibition kinetics and primer-extension analyses determined biochemical mechanisms of FIAU, y-2-fluoro-beta-D-arabinofuranosyl)-5-methyluraci l (FAU), and 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)uracil tripho sphate (TP) inhibition of DNA pol-gamma, dTMP incorporation by DNA pol -gamma was inhibited competitively by FIAUTP, FMAUTP, and FAUTP (K-i = 0.015, 0.03, and 1.0 mu M, respectively), By using oligonucleotide te mplate-primers, DNA pol-gamma incorporated each analog into DNA opposi te a single adenosine efficiently without effects on DNA chain elongat ion, Incorporation of multiple adjacent analogs at positions of consec utive adenosines dramatically impaired chain elongation by DNA pol-gam ma, Effects of FIAU, FMAU, and FAU on HepG2 cell mtDNA abundance and u ltrastructure were determined, After 14 days, mtDNA decreased by 30% w ith 20 mu M FIAU or 20 mu M FMAU and decreased less than 10% with 100 mu M FAU, FIAU and FMAU disrupted mitochondria and caused accumulation of intracytoplasmic lipid droplets, Biochemical and cell biological f indings suggest that FIAU and its metabolites inhibit mtDNA replicatio n, most likely at positions of adenosine tracts, leading to decreased mtDNA and mitochondrial ultrastructural defects.