Cellular level of 8-oxo-2'-deoxyguanosine in DNA does not correlate with urinary excretion of the modified base/nucleoside (original) (raw)
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Methods in molecular biology (Clifton, N.J.), 2011
The ability to non-invasively assess DNA oxidation and its repair, has significant utility in large-scale, population-based studies. Such studies could include the assessments of: the efficacy of antioxidant intervention strategies, pathological roles of DNA oxidation in various disease states and population or interindividual differences in antioxidant defence and DNA repair. The most popular method, to non-invasively assess oxidative insult to the genome is by the analysis of urine for 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), using chromatographic techniques or immunoassay procedures. The provenance of extracellular 8-oxodG remains a subject for debate. However, previous studies have shown that factors, such as diet and cell death, do not appear to contribute to extracellular 8-oxodG, leaving processes, such as the repair of DNA and/or the 2'-deoxyribonucleotide pool, as the sole source of endogenous 8-oxodG. The method in this chapter describes a non-invasive approa...
Comparison of different methods of measuring 8-oxoguanine as a marker of oxidative DNA damage
Free Radical Research, 2000
We are attempting to resolve some of the problems encountered in measuring 8-hydroxy-2'-deoxyguanosine (8-oxodG) in human cellular DNA as a marker of oxidative stress. Samples of authentic 8-oxodG were distributed, and participating laboratories undertook to analyse this material within a specified period. Most HPLC procedures gave values for 8-oxodG within :t:40% of the target, as did two of four GC-MS procedures, and both LC-MS-MS methods. Calf thymus DNA samples containing increasing amounts of 8-oxodG were also distributed for analysis. Fewer than half the procedures tested were able to detect the dose response; those that were successful tended to be procedures with low coefficients of variation. For the analysis of 8-oxodG in human cells, where it is likely to be present at much lower concentrations than in the calf thymus DNA, it is crucial to reduce analytical variation to a minimum; a coefficient of variation of less than 10% should be the aim, to give reasonable precision. HPLC with amperometric electrochemical detection is not recommended, as it is less sensitive than coulometric detection. Immunological detection, 32p-postlabelling and LC-MS-MS are alternative approaches to measurement of 8-oxodG in DNA that, on the grounds of precision ~ and detection of dose response, cannot at present be recommended.
Antioxidants & Redox Signaling, 2006
Numerous DNA repair pathways exist to prevent the persistence of damage, and are integral to the maintenance of genome stability, and hence prevention of disease. Excised lesions arising from repair may ultimately appear in the urine where their measurement has been acknowledged to be reflective of overall oxidative stress. The development of reliable assays to measure urinary DNA lesions, such as HPLC prepurification followed by gas chromatography/mass spectrometry, offers the potential to assess whole body oxidative DNA damage. However, some studies suggest a possibility that confounding factors may contribute to urinary lev-
Levels of 8-hydroxydeoxyguanosine as a marker of DNA damage in human leukocytes
Free Radical Biology and Medicine, 2000
We measured 8-hydroxy-2-deoxyguanosine (8-OHdG) levels in human leukocytes from healthy donors to evaluate oxidative DNA damage and its correlation with smoking, physical exercise, and alcohol consumption. A significant increase in oxidative DNA damage was induced by cigarette smoke, with the mean level of 8-OHdG being significantly higher in smokers (33.1 Ϯ 10.6 per 10 6 2-deoxyguanosine (dG) [mean Ϯ SE], n ϭ 16) compared with nonsmokers (15.3 Ϯ 1.8 per 10 6 dG, n ϭ 31) and former smokers (17.8 Ϯ 1.5 per 10 6 dG, n ϭ 9). The highest values were observed after smoking more than 10 cigarettes per day (41.8 Ϯ 17.1 per 10 6 dG, n ϭ 9). A large interindividual variation in 8-OHdG levels was observed in all analyzed groups. We also observed a correlation between 8-OHdG levels and age in nonsmokers and former smokers. Neither frequency of physical exercise nor alcohol drinking significantly modified 8-OHdG levels in leukocytes.
European Journal of Cancer, 1995
An automated analytical method has been developed for determination of the oxidative DNA adduct, Bhydroxydeoxyguanosine (80HdG) in human urine, based on coupled-column high performance liquid chromatography with electrochemical detection. Urine is concentrated on Bondelut CH by means of an automated sample processor, and the enriched sample injected on to a polymeric reversed phase column coupled in line with an electrochemical detector and a C,, reversed phase column. By use of the electrochemical detector, a suitable retention time interval is set for collection of the fraction containing 80HdG from the chromatography on the first column; this fraction is collected in a 2 mL loop and injected onto the Cls column. The system is operated by an automatic valve station controlled by an integrator. The method has a large sample capacity and measures 31.1, 15.7, and 7.43 nmol BOHdG/L urine with variation coefficients of 8, 8 and 24% within series and 8, 11 and 23% between series. Normal healthy individuals were found to excrete 14.9 ? 7.8 nmol 80HdG/24 h, or 1.11-+ 0.62 pmol80HdG per mol creatinine, in their urine, whereas increased levels of 80HdG were found in 24 h collections from a variety of cancer patients, both in samples taken before onset of oncological therapy (1.84 + 1.12 pmohmol creatinine, P < 0.01 versus healthy individuals) and after therapy onset (2.18-C 1.44 pmol/mol creatinine, P < 0.001 versus healthy individuals). Moreover, mean values of 80HdG in random urinary samples from cancer patients were significantly higher than from healthy individuals (2.42 2 2.28 versus 1.19 2 0.48 pmohmol creatinine, P < O.OOl), both in samples taken before therapy onset (1.91 2 0.96, P < 0.001 versus healthy individuals) and after (2.57 * 2.46, P < 0.001 versus healthy individuals). High levels of urinary 80HdG were found in patients subjected to whole body irradiation, and in patients receiving chemotherapy with various cytostatic agents. The potential use of the method for detecting increased urinary 80HdG excretion and conditions associated with increased oxidative DNA damage in humans is discussed.
Carcinogenesis, 2002
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-7,8dihydro-2Ј-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 Ooxidized 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 beststudied protocol, the steady-state cellular background level of 8-oxodGuo, in a lymphocyte cell line, was determined to be~0.5 lesions/10 6 DNA nucleosides.
Analysis of 7,8Dihydro8-oxo-2′-deoxyguanosine in Cellular DNA during Oxidative Stress
Chemical Research in Toxicology, 2009
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 chromatographybased assays. H358 cells were treated with increasing concentrations of potassium bromate (KBrO 3 ) 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 KBrO 3 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-7,8dione 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. * Corresponding
Nucleic Acids Research, 2001
DNase I, phosphodiesterases I and II and alkaline phosphatase. The atmospheric pressure ionizationelectrospray process was used for mass spectral measurements. A stable isotope-labeled analog of 8-OH-dGuo was used as an internal standard for quantification by isotope-dilution MS (IDMS). Results showed that LC/IDMS with selected ion-monitoring (SIM) is well suited for identification and quantification of 8-OH-dGuo in DNA at background levels and in damaged DNA. The sensitivity level of LC/IDMS-SIM was found to be comparable to that reported previously using LC-tandem MS (LC/MS/MS). It was found that approximately five lesions per 10 6 DNA bases can be detected using amounts of DNA as low as 2 µg. The results also suggest that this lesion may be quantified in DNA at levels of one lesion per 10 6 DNA bases, or even lower, when more DNA is used. Up to 50 µg of DNA per injection were used without adversely affecting the measurements. Gas chromatography/ isotope-dilution MS with selected-ion monitoring (GC/ IDMS-SIM) was also used to measure this compound in DNA following its removal from DNA by acidic hydrolysis or by hydrolysis with Escherichia coli Fpg protein. The background levels obtained by LC/IDMS-SIM and GC/IDMS-SIM were almost identical. Calf thymus DNA and DNA isolated from cultured HeLa cells were used for this purpose. This indicates that these two techniques can provide similar results in terms of the measurement of 8-OH-dGuo in DNA. In addition, DNA in buffered aqueous solution was damaged by ionizing radiation at different radiation doses and analyzed by LC/IDMS-SIM and GC/IDMS-SIM. Again, similar results were obtained by the two techniques. The sensitivity of GC/MS-SIM for 7,8dihydro-8-oxoguanine was also examined and found to be much greater than that of LC/MS-SIM and the reported sensitivity of LC/MS/MS for 8-OH-dGuo. Taken together, the results unequivocally show that LC/IDMS-SIM is well suited for sensitive and accurate measurement of 8-OH-dGuo in DNA and that both LC/ IDMS-SIM and GC/IDMS-SIM can provide similar results.