Fluorescence detection of 8-oxoguanine in nuclear and mitochondrial DNA ofcultured cells using a recombinant Fab and confocal scanning laser microscopy

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.