Analysis of 7,8-dihydro-8-oxo-2'-deoxyguanosine in cellular DNA during oxidative stress

Abstract

Analysis of cellular 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo) as a biomarker of oxidative DNA damage has been fraught with numerous methodological problems. This is primarily due to artifactual oxidation of dGuo that occurs during DNA isolation and hydrolysis. Therefore, it has become necessary to rely on using the comet assay, which is not necessarily specific for 8-oxo-dGuo. A highly specific and sensitive method based on immunoaffinity purification and stable isotope dilution liquid chromatography (LC)-multiple reaction monitoring (MRM)/mass spectrometry (MS) that avoids artifact formation has now been developed. Cellular DNA was isolated using cold DNAzol (a proprietary product that contains guanidine thiocyanate) instead of chaotropic- or phenol-based methodology. Chelex-treated buffers were used to prevent Fenton chemistry-mediated generation of reactive oxygen species (ROS) and artifactual oxidation of DNA bases. Deferoxamine was also added to all buffers in order to complex any residual transition metal ions remaining after Chelex treatment. The LC-MRM/MS method was used to determine that the basal 8-oxo-dGuo level in DNA from human bronchoalveolar H358 cells was 2.2 +/- 0.4 8-oxo-dGuo/10(7) dGuo (mean +/- standard deviation) or 5.5 +/- 1.0 8-oxo-dGuo/10(8) nucleotides. Similar levels were observed in human lung adenocarcinoma A549 cells, mouse hepatoma Hepa-1c1c7 cells, and human HeLa cervical epithelial adenocarcinoma cells. These values are an order of magnitude lower than is typically reported for basal 8-oxo-dGuo levels in DNA as determined by other MS- or chromatography-based assays. H358 cells were treated with increasing concentrations of potassium bromate (KBrO3) as a positive control or with the methylating agent methyl methanesulfonate (MMS) as a negative control. A linear dose-response for 8-oxo-dGuo formation (r(2) = 0.962) was obtained with increasing concentrations of KBrO3 in the range of 0.05 mM to 2.50 mM. In contrast, no 8-oxo-dGuo was observed in H358 cell DNA after treatment with MMS. At low levels of oxidative DNA damage, there was an excellent correlation between a comet assay that measured DNA single strand breaks (SSBs) after treatment with human 8-oxo-guanine glycosylase-1 (hOGG1) when compared with 8-oxo-dGuo in the DNA as measured by the stable isotope dilution LC-MRM/MS method. Availability of the new LC-MRM/MS assay made it possible to show that the benzo[a]pyrene (B[a]P)-derived quinone, B[a]P-7,8-dione, could induce 8-oxo-dGuo formation in H358 cells. This most likely occurred through redox cycling between B[a]P-7,8-dione and B[a]P-7,8-catechol with concomitant generation of DNA damaging ROS. In keeping with this concept, inhibition of catechol-O-methyl transferase (COMT)-mediated detoxification of B[a]P-7,8-catechol with Ro 410961 caused increased 8-oxo-dGuo formation in the H358 cell DNA.

Scheme 1. B[a]P-7,8-dione-Mediated Formation of 8-Oxo-dGuo in H358 Cells

Figure 1

Formation of 8-oxo-dGuo in human bronchoalveolar H358 cell DNA using different isolation and hydrolysis methods. DNAzol isolation is shown as solid bars and NaI as slanted striped bars. Analyses were conducted by immunoaffinity purification and stable isotope dilution LC-MRM/MS analysis of 8-oxo-dGuo in H358 cell hydrolysate (10⁶ cells) using Chelex-treated buffers in the absence or presence of deferoxamine or TEMPO chelators. Data are presented as means ± SD (error bars) from triplicate samples. P values were determined by an unpaired Student’s t-test.

Figure 2

Immunoaffinity purification coupled with stable isotope dilution LC-MRM/MS analysis of 8-oxo-dGuo isolated from H358 cell DNA using the DNAzol method with deferoxamine to chelate transition metal ions. The upper panel shows the MRM chromatogram for endogenous 8-oxo-dGuo (2.8 8-oxo-dGuo/10⁷ dGuo), and the lower panel shows the internal standard’s chromatogram.

Figure 3

Linear relationship between DMSO treatment and 8-oxo-dGuo formation. DMSO-mediated 8-oxo-dGuo formation in H358 cells was determined by LC-MRM/MS. Data are presented as means ± SD (error bars) from duplicate samples.

Figure 4

Linear relationship between KBrO₃ treatment and 8-oxo-dGuo formation. KBrO₃-mediated 8-oxo-dGuo formation in H358 cells was determined by LC-MRM/MS. Data are presented as means ± SD (error bars or within symbols) from duplicate samples.

Figure 5

MMS-mediated 8-oxo-dGuo formation in H358 cells as determined by LC-MRM/MS (●), comet assay with hOGG1 (◼), or comet assay without hOGG1 (▼). Data are presented as means ± SD (error bars or within symbols) from duplicate samples.

Figure 6

KBrO₃-mediated 8-oxo-dGuo formation in H358 cells as determined by the comet assay with hOGG1 (◼) or the comet assay without hOGG1 (▼). Data are presented as means ± SD (error bars or within symbols) from duplicate samples.

Figure 7

Analysis of 8-oxo-dGuo in H358 cells by LC-MRM/MS after treatment with PBS (right slanted stripes), 1% DMSO vehicle (open), B[a]P-7,8-dione (left slanted stripes), 3 μM COMT inhibitor Ro 410961 (shaded), and 3 μM Ro 410961 + 20 μM B[a]P-7,8-dione (basket weave). Data are presented as means ± SD (error bars) from triplicate samples. P values were determined by an unpaired Student’s t-test.