Artifactual formation of 8-oxo-2'-deoxyguanosine: role of fluorescent light and inhibitors.
The pro-mutagenic oxidative DNA lesion, 8-oxo-2'-deoxyguanosine (8-oxodG) has been a subject of numerous studies. However, the absolute 8-oxodG levels in tissue DNA reported by various methods have been debated due to its artifactual production during DNA isolation and/or the DNA processing. We have investigated factors that may result in such artifacts during isolation and analysis of DNA as well as means for its prevention. 8-OxodG content was measured by a recently described TLC enrichment-mediated 32P-postlabeling. Liver DNA from 3 month-old, female Sprague-Dawley rats was isolated by a standard solvent-extraction procedure (phenol, phenol:Sevag, and Sevag; 23 degrees C), a modified solvent-extraction procedure (phenol:Sevag, and Sevag; 4 degrees C; KCl-SDS-protein precipitation) or sodium iodide extraction procedure. 8-OxodG was analyzed in the DNA by the 32P-postlabeling assay using a fluorescent light box during the workup, as well as in its absence. The 8-oxodG levels, when the fluorescent light box was used, were in similar range irrespective of the DNA isolation procedure (16.4+/-1.6 to 28.7+/-6 8-oxodG/10(6) nucleotides). However, the values were significantly lower (3.1+/-0.4 to 3.4+/-0.2 8-oxodG/10(6) nucleotides) in the absence of fluorescence light box, room fluorescent light (suspended through the ceiling) and natural room light did not alter the 8-oxodG levels. Further, the addition of 0.3 mM of PBN (N-t-butyl-alpha-phenyl nitrone) or TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl), or 6.8 mM 8-hydroxy-quinoline during the DNA isolation resulted in still lower values (0.8+/-0.1 to 1.8+/-0.5 8-oxodG/10(6) nucleotides) although this reduction was not consistently observed in different experiments. These results suggest that fluorescent light is the major 'culprit' in artifactual production and variability reported in the 8-oxodG levels.
Arif
Jamal M
JM
Biological and Medical Research, King Faisal Specialist Hospital and Research Center, Riyadh 11211, Saudi Arabia.
Gupta
Ramesh C
RC
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D002855:Chromatography, Thin Layer; D004247:DNA / Q000737:chemistry / Q000528:radiation effects*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000494:pharmacology*; D005260:Female; D005453:Fluorescence; D008027:Light; D008099:Liver / Q000378:metabolism / Q000528:radiation effects; D009153:Mutagens; D051381:Rats; D017207:Rats, Sprague-Dawley; D012974:Sodium Iodide / Q000494:pharmacology
D009153:Mutagens; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D012974:Sodium Iodide; D003849:Deoxyguanosine