Abstract
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.
| Original language | English |
|---|---|
| Pages (from-to) | 199-212 |
| Number of pages | 14 |
| Journal | Methods in molecular biology (Clifton, N.J.) |
| Volume | 554 |
| DOIs | |
| Publication status | Published - Jan 1 2009 |
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All Science Journal Classification (ASJC) codes
- Molecular Biology
- Genetics
Cite this
Quantitative analysis of oxidized guanine, 8-oxoguanine, in mitochondrial DNA by immunofluorescence method. / Mizuki, Ohno; Oka, Sugako; Nakabeppu, Yusaku.
In: Methods in molecular biology (Clifton, N.J.), Vol. 554, 01.01.2009, p. 199-212.Research output: Contribution to journal › Special issue
}
TY - JOUR
T1 - Quantitative analysis of oxidized guanine, 8-oxoguanine, in mitochondrial DNA by immunofluorescence method.
AU - Mizuki, Ohno
AU - Oka, Sugako
AU - Nakabeppu, Yusaku
PY - 2009/1/1
Y1 - 2009/1/1
N2 - 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.
AB - 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.
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UR - http://www.scopus.com/inward/citedby.url?scp=70349655929&partnerID=8YFLogxK
U2 - 10.1007/978-1-59745-521-3_13
DO - 10.1007/978-1-59745-521-3_13
M3 - Special issue
C2 - 19513676
AN - SCOPUS:70349655929
VL - 554
SP - 199
EP - 212
JO - Methods in Molecular Biology
JF - Methods in Molecular Biology
SN - 1064-3745
ER -