[32P]ATP mediates formation of 8-hydroxy-2'-deoxyguanosine from 2'-deoxyguanosine, a possible problem in the 32P-postlabeling assay.

Abstract

8-Hydroxy-2'-deoxyguanosine (8-OH-dG) is a biomarker for oxidative stress on DNA, a common lesion in mammalian cells. A correlation between increased levels of 8-OH-dG and diseases like diabetes, infections and cystic fibrosis has been found in humans. 8-OH-dG levels have been shown to be decreased by antioxidants, an indication of the importance of dietary habits. 8-OH-dG is used as a biomarker for oxidative stress in vivo as well as in vitro and is suggested to be a mutagenic DNA lesion. Different methods are used for the analyses of 8-OH-dG, i.e. GC-MS, HPLC-EC and 32P-postlabeling. The most commonly used method is HPLC-EC. In the analysis of 8-OH-dG, the work-up procedure for DNA, as well as the preparation for analysis, are of critical importance as there is a risk for auto-oxidation of deoxyguanosine (dG), which would result in false high background levels and low sensitivity in analysis. 32P-Postlabeling has recently been applied to the analysis of 8-OH-dG and has shown to be a very sensitive method for the detection of DNA adducts. It is shown here that after extrapolation to normal 32P-postlabeling conditions, [32P]ATP generated 8-OH-dG to levels of 25 8-OH-dG/10(5) dG. [32P]ATP mediated the formation of 8-OH-dG from dG in a dose-dependent manner at all dose levels (0.13-12 microCi). The reaction occurred immediately and increased with time in a linear dose-response fashion. At high doses (6.0 and 12 microCi) the dose-response declined after 24 h, which indicates a possible decomposition or rearrangement of 8-OH-dG. A repeated experiment with 5 microCi [32P]ATP during 2 h resulted in a linear formation of 8-OH-dG and a level of 19 8-OH-dG/10(5) dG. The results indicated that awareness of the auto-oxidation generated by 32P[ATP] in the postlabeling assay is of utmost importance and that dG must be separated before 32P-postlabeling of 8-OH-dG.

• Authors: L Möller, T Hofer
• Journal: Carcinogenesis (Carcinogenesis) Vol. 18 Issue 12 Pg. 2415-9 (Dec 1997) ISSN: 0143-3334 [Print] England
• PMID: 9450489 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Phosphorus Radioisotopes
• 8-Hydroxy-2'-Deoxyguanosine
• Adenosine Triphosphate
• DNA
• Deoxyguanosine

Topics

• 8-Hydroxy-2'-Deoxyguanosine
• Adenosine Triphosphate (chemistry)
• DNA (chemistry)
• DNA Damage
• Deoxyguanosine (analogs & derivatives, chemistry, radiation effects)
• Oxidation-Reduction
• Phosphorus Radioisotopes