Cellular background level of 8-oxo-7,8-dihydro-2'-deoxyguanosine: an isotope based method to evaluate artefactual oxidation of DNA during its extraction and subsequent work-up

Abstract

The measurement of oxidative damage to cellular DNA is a challenging analytical problem requiring highly sensitive and specific methods. In addition, artefactual DNA oxidation during its extraction and subsequent work-up may give rise to overestimated levels of oxidized DNA bases. In the present study, we have used (18)O-labelled 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) as an internal standard to evaluate the extent of artefactual DNA oxidation during the critical steps preceding the measurement. The labelled oxidized purine nucleoside was specifically generated in cellular DNA using the recently available generator of (18)O-labelled singlet oxygen. Artefactual DNA oxidation that could take place during the work-up increases the level of 8-oxodGuo but not of the (18)O-oxidized nucleoside. Therefore, the ratio between the two compounds, as measured by high performance liquid chromatography coupled to tandem mass spectrometry, allows an unambiguous comparison of different methodologies. The comparison of different DNA extraction protocols led to the conclusion that artefactual DNA oxidation during the extraction step could be minimized if: (i) nuclei are isolated after cell lysis; (ii) desferrioxamine, a transition metal chelator is added to the different extraction buffers; and (iii) sodium iodide (or alternatively guanidine thiocyanate) is used for DNA precipitation. It was also demonstrated that sodium iodide does not decompose the targeted oxidized purine nucleoside. In addition, three different DNA digestion protocols were evaluated and they were found to give rise to similar results. Using the best-studied protocol, the steady-state cellular background level of 8-oxodGuo, in a lymphocyte cell line, was determined to be approximately 0.5 lesions/10(6) DNA nucleosides.

HPLC Measurement of the DNA Oxidation Biomarker, 8-oxo-7,8-dihydro-2’-deoxyguanosine, in Cultured Cells and Animal Tissues

“…A possibility that incomplete DNA digestion resulted in fragments that are too small to be detected on the agarose gel remains. Even though such a systematic error would affect all samples equally, further experiments (e.g., treatment of cultured cells with a radiolabelled pro-oxidant 10 ) could be conducted to completely rule out such a possibility.…”

“…Remove PBS and store the cell pellet at -80 °C until further analysis. Artificial DNA oxidation can be further minimized by nuclear isolation, as described elsewhere 10 .…”

Oxidation of the sugar moiety of DNA by ionizing radiation or bleomycin could induce the formation of a cluster DNA lesion

“…Formation of 4 was also detected upon ␥-irradiation of 2.5 mM 3Ј-TMP (50 Gy) subsequently incubated in the presence of dCyd, without any enzymatic digestion. DNA was extracted from THP1 cells, a human monocyte cell line, by using a recently optimized protocol (29).…”

UVA radiation is highly mutagenic in cells that are unable to repair 7,8-dihydro-8-oxoguanine in Saccharomyces cerevisiae

“…Upon UVA irradiation, rad14 ogg1 double-mutant cells were harvested, and genomic DNA was immediately extracted in the presence of 0.1 mM desferrioxamine mesylate (Sigma) to prevent the formation of 8-oxoG during the procedure (29). Aliquots (10 g) of DNA from irradiated or nonirradiated cells were incubated for 45 min at 37°C in a reaction buffer (12-l final volume) that contained 250 ng of Escherichia coli Fpg protein (our laboratory stock) or 1,000 units of T4 denV protein (Trevigen, Gaithersburg, MD).…”