Fluorescence detection of 8-oxoguanine in nuclear and mitochondrial DNA ofcultured cells using a recombinant Fab and confocal scanning laser microscopy
Rp. Soultanakis et al., Fluorescence detection of 8-oxoguanine in nuclear and mitochondrial DNA ofcultured cells using a recombinant Fab and confocal scanning laser microscopy, FREE RAD B, 28(6), 2000, pp. 987-998
The presence of 8-oxoguanine (8-oxoG) in DNA is considered a marker of oxid
ative stress and DNA damage. We describe a multifluorescence technique to d
etect the localization of 8-oxoG in both nuclear and mitochondrial DNA usin
g a mouse recombinant Fab 166. The Fab was generated by repertoire cloning
and combinatorial phage display, and specifically recognized 8-oxoG in DNA,
as determined by competitive enzyme-linked immunosorbent assays (ELISAs).
In situ detection of 8-oxoG was accomplished using rat lung epithelial (RLE
) cells and human B lymphoblastoid (TK6) cells treated with hydrogen peroxi
de (H2O2) or ionizing radiation, respectively. Using confocal scanning lase
r microscopy, we observed nuclear and perinuclear immunoreactivity of 8-oxo
G in control cultures. The simultaneous use of a nuclear DNA stain, propidi
um iodide, or the mitochondrial dye, MitoTracker (Molecular Probes, Eugene,
OR, USA), confirmed that 8-oxoG immunofluorescence occurred in nuclear and
mitochondrial DNA. Marked increases in the presence of 8-oxoG in nuclear D
NA were apparent after treatment with H2O2 or ionizing radiation. In contro
l experiments, Fab 166 was incubated with 200 mu M purified 8-oxodG or with
formamidopyrimidine DNA-glycosylase (Fpg) to remove 8-oxoG lesions in DNA.
These protocols attenuated both nuclear and mitochondrial staining. We con
clude that both nuclear and mitochondrial oxidative DNA damages can be simu
ltaneously detected in situ using immunofluorescence labeling with Fab 166
and confocal microscopy. (C) 2000 Elsevier Science Inc.