Preferential mitochondrial DNA injury caused by glucose oxidase as a steady generator of hydrogen peroxide in human fibroblasts.
To test the hypothesis that mitochondrial DNA (mtDNA) is more prone to reactive oxygen species (ROS) damage than nuclear DNA, a continuous flux of hydrogen peroxide (H2O2) was produced with the glucose/glucose oxidase system. Using a horse radish peroxidase (HRPO)-based colorimetric assay to detect H2O2, glucose oxidase (GO; 12 mU/ml) produced 95 microM of H2O2 in 1 h, whereas only 46 microM of hydrogen peroxide accumulated in the presence of SV40-transformed human fibroblasts ( approximately 1 x 10(6). DNA damage was assessed in the mitochondira and three nuclear regions using a quantitative PCR assay. GO (12 mU/ml) resulted in more damage to the mitochondrial DNA (2.250 +/- 0.045 lesions/10 kb) than in any one of three nuclear targets, which included the non-expressed beta-globin locus (0.436 +/- 0.029 lesions/10 kb); and the active DNA polymerase b gene (0.442 +/- 0.037 lesions/10 kb); and the active hprt gene (0.310 +/- 0.025). Damage to the mtDNA occurred within 15 min of GO treatment, whereas nuclear damage did not appear until after 30 min, and reached a maximum after 60 min. Repair of mitochondrial damage after a 15 min GO (6 mU/ml) treatment was examined. Mitochondria repaired 50% of the damage after 1 h, and by 6 h all the damage was repaired. Higher doses of GO-generated H202, or more extended treatment periods, lead to mitochondrial DNA damage which was not repaired. Mitochondrial function was monitored using the MTT (3,(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) assay. A 15 min treatment with 6 mU/ml of GO decreased mitochondrial activity to 80% of the control; the activity recovered completely within 1 h after damage. These data show that GO-generated H202 causes acute damage to mtDNA and function, and demonstrate that this organelle is an important site for the cellular toxicity of ROS.
Salazar
J J
JJ
Sealy Center for Molecular Science, University of Texas Medical Branch, Galveston 77555, USA.
Van Houten
B
B
D002461:Cell Line, Transformed; D004247:DNA / Q000032:analysis; D004249:DNA Damage; D019951:DNA Polymerase beta / Q000235:genetics; D004260:DNA Repair; D004272:DNA, Mitochondrial / Q000235:genetics*; D005347:Fibroblasts; D005914:Globins / Q000235:genetics; D005949:Glucose Oxidase / Q000494:pharmacology*; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism*; D007041:Hypoxanthine Phosphoribosyltransferase / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology; D013385:Succinate Dehydrogenase / Q000378:metabolism
D004272:DNA, Mitochondrial; D005914:Globins; D004247:DNA; D006861:Hydrogen Peroxide; D005949:Glucose Oxidase; D013385:Succinate Dehydrogenase; D007041:Hypoxanthine Phosphoribosyltransferase; D019951:DNA Polymerase beta