Quantitative analysis of oxidized guanine, 8-oxoguanine, in mitochondrial DNA by immunofluorescence method.
8-Oxoguanine (8-oxoG), an oxidized form of guanine, is one of the major mutagenic lesions generated under oxidative stress. Oxidative damage in mitochondrial DNA has been implicated as a causative factor for a wide variety of degenerative diseases as well as for cancer during aging. We established a quantitative method for in situ detection of 8-oxoG in mitochondrial DNA in a single-cell level using a monoclonal antibody. Specific detection of 8-oxoG in mitochondrial DNA was confirmed by pre-treatment of samples with DNase I or MutM, the latter excising 8-oxoG opposite C in DNA. We then analyzed 8-oxoG dynamics in mitochondrial DNA of the wild-type and 8-oxoG DNA glycosylase (OGG1)-deficient mouse cells after exposure to hydrogen peroxide. Intensities for the 8-oxoG immunoreactivity in mitochondrial DNA were increased immediately after the exposure to hydrogen peroxide in both types of cells. The increased intensities returned to basal levels within a few hours only in wild-type cells, but not in OGG1-deficient cells which exhibited the increased intensities even 24 h after the exposure. These results indicate that OGG1 is a major enzyme for excision repair of 8-oxoG in mitochondrial DNA in mouse cells, and that our method described here is appropriate to study 8-oxoG dynamics in mitochondrial DNA.
Ohno
Mizuki
M
Division of Neurofunctional Genomics, Department of Immunology and Neuroscience, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan.
Oka
Sugako
S
Nakabeppu
Yusaku
Y
D000818:Animals; D001921:Brain / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D045647:DNA Glycosylases / Q000502:physiology; D004272:DNA, Mitochondrial / Q000737:chemistry*; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D005455:Fluorescent Antibody Technique / Q000379:methods*; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000494:pharmacology; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000378:metabolism; D041681:NIH 3T3 Cells; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction
D004272:DNA, Mitochondrial; D016877:Oxidants; C024829:8-hydroxyguanine; D006147:Guanine; D006861:Hydrogen Peroxide; D045647:DNA Glycosylases; C485990:Ogg1 protein, mouse