Biological role of 8-oxo-2'-deoxyguanosine

The review presents currently available data on the biological role of 8-oxo-2’-deoxyguanosine. This compound for a long time has been used successfully as a biomarker of oxidative stress and diseases associated with it. However, in recent years, an increasing number of publications has appeared reporting that biological role of 8-oxo-dG is not only a mutagenic one; it also is involved in the regulation of gene expression, in some processes of DNA repair, in the control of inflammatory and autoimmune reactions, and in the activation of antioxidant systems. Probably, 8-oxo-2’-deoxyguanosine could prospectively be used as a drug.     

Simultaneous measurement of 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine by HPLC-MS/MS

An assay with high selectivity and sensitivity has been developed which, for the first time, allows quantitative, simultaneous measurement in DNA of both 8-oxo-2'-deoxyguanosine (8-oxodG) and 8-oxo-2'-deoxyadenosine (8-oxodA)-important biomarkers of oxidative DNA damage in vivo. Using reversed-phase HPLC coupled to electrospray tandem mass spectrometry (HPLC-MS/MS) in multiple reaction monitoring (MRM) mode it was possible to detect background levels of these lesions in commercially available calf thymus DNA (85 +/- 3 and 7.1 +/- 0.2 per 10(6) DNA bases for 8-oxodG and 8-oxodA respectively; n = 3). Levels of 8-oxodG determined by HPLC coupled to an electrochemical detection system (HPLC-EC) were found to be similar (75 +/- 6 per 10(6) DNA bases; n = 3) to those obtained using tandem mass spectrometry.     

Influence of DNA torsional rigidity on excision of 7,8-dihydro-8-oxo-2'-deoxyguanosine in the presence of opposing abasic sites by human OGG1 protein

The human protein OGG1 (hOGG1) targets the highly mutagenic base 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) and shows a high specificity for the opposite DNA base. Abasic sites can arise in DNA in close opposition to 8-oxodG either during repair of mismatched bases (i.e. 8-oxodG/A mismatches) or, more frequently, as a consequence of ionizing radiation exposure. Bistranded DNA lesions may remain unrepaired and lead to cell death via double-strand break formation. In order to explore the role of damaged-DNA dynamics in recognition/excision by the hOGG1 repair protein, specific oligonucleotides containing an 8-oxodG opposite an abasic site, at different relative distances on the complementary strand, were synthesized. Rotational dynamics were studied by means of fluorescence polarization anisotropy decay experiments and the torsional elastic constant as well as the hydrodynamic radius of the DNA fragments were evaluated. Efficiency of excision of 8-oxodG was tested using purified human glycosylase. A close relation between the twisting flexibility of the DNA fragment and the excision efficiency of the oxidative damage by hOGG1 protein within a cluster was found.     

OGG1 mRNA expression and incision activity in rats are higher in foetal tissue than in adult liver tissue while 8-oxo-2'-deoxyguanosine levels are unchanged

This study was set up to investigate the relationships between the formation and removal of DNA damage in form of 8-oxodeoxyguanosine (8-oxodG) in neonatal (day 16 of gestation) as compared to adult rats. The hypothesis addressed was whether the rapidly dividing foetal tissue has an enhanced requirement of DNA repair providing protection against potentially mutagenic DNA damages such as 8-oxodG. The activity of the primary 8-oxodG-repair protein OGG1 was measured by a DNA incision assay and the expression of OGG1 mRNA was measured by Real-Time PCR normalised to 18S rRNA. The tissue level of 8-oxodG was measured by HPLC-ECD. We found a 2-3-fold increased incision activity in the foetal control tissue, together with a 3-15-fold increase in mRNA of OGG1 as compared to liver tissue from adult rats. The levels of 8-oxodG in the foetal tissue were unaltered as compared to the adult groups. To increase the levels of 8-oxodG, the rats received an injection (i.p.) of the hepatotoxin 2-nitropropane. The compound induced significant levels of 8-oxodG in male rat livers 5h after the injection and in the foetuses 24h after the injection, while the female rats showed no increase in 8-oxodG. The incision activity was slightly depressed in both male and female liver tissue and in the foetal tissue 5h after the injection, but significantly increased from 5 to 24h after the injection. However, it did not reach levels significantly above the control levels.In conclusion, this study confirms that foetal tissue has increased levels of OGG1 mRNA and correspondingly an enhanced incision activity on an 8-oxodG substrate in a crude tissue extract.     

Inter-laboratory validation of procedures for measuring 8-oxo-7,8-dihydroguanine/8-oxo-7,8-dihydro-2'-deoxyguanosine in DNA

The aim of ESCODD, a European Commission funded Concerted Action, is to improve the precision and accuracy of methods for measuring 8-oxo-7,8-dihydroguanine (8-oxoGua) or the nucleoside (8-oxodG). On two occasions, participating laboratories received samples of different concentrations of 8-oxodG for analysis. About half the results returned (for 8-oxodG) were within 20% of the median values. Coefficients of variation (for three identical samples) were commonly around 10%. A sample of calf thymus DNA was sent, dry, to all laboratories. Analysis of 8-oxoGua/8-oxodG in this sample was a test of hydrolysis methods. Almost half the reported results were within 20% of the median value, and half obtained a CVof less than 10%. In order to test sensitivity, as well as precision, DNA was treated with photosensitiser and light to introduce increasing amounts of 8-oxoGua and samples were sent to members. Median values calculated from all returned results were 45.6 (untreated), 53.9, 60.4 and 65.6 8-oxoGua/10(6) Gua; only seven laboratories detected the increase over the whole range, while all but one detected a dose response over two concentration intervals. Results in this trial reflect a continuing improvement in precision and accuracy. The next challenge will be the analysis of 8-oxodG in DNA isolated from cells or tissue, where the concentration is much lower than in calf thymus DNA.     

Epigallocatechin gallate markedly enhances formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in the reaction of 2'-deoxyguanosine with hypochlorous acid

A tea polyphenol, (-)-epigallocatechin gallate (EGCG), which can scavenge a variety of reactive oxygen species, enhances the yield of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) up to 20-fold in the reaction of 2'-deoxyguanosine with hypochlorous acid (HOCl), compared with the reaction without EGCG. Certain concentrations of EGCG inhibited HOCl-mediated oxidation of 2'-deoxyguanosine to 8-oxo-dG to a limited extent, but efficiently inhibited further oxidation of 8-oxo-dG to spiroiminodihydantoin nucleoside, resulting in the accumulation of 8-oxo-dG in the reaction mixture. Conversely, EGCG inhibited dose-dependently an increase in 8-oxo-dG levels in calf thymus DNA incubated with HOCl. However, addition of HOCl to the DNA preoxidized with an oxidant-generating system (CuCl2, ascorbate, H2O2), led to the extensive loss of 8-oxo-dG due to its further oxidation. EGCG effectively inhibited this HOCl-mediated loss of 8-oxo-dG in the oxidized DNA, resulting in an apparent increase in 8-oxo-dG levels in the oxidized DNA, compared with the levels found without EGCG. The conversion of 8-oxo-dG into other oxidized lesions will inevitably affect recognition by DNA repair enzymes as well as the rates of mutations and DNA synthesis. Thus, our results suggest that as a biomarker of oxidative DNA damage, not only 8-oxo-dG but also the products of its further oxidation should be analyzed.     

Leukocyte 8-oxo-7,8-dihydro-2'-deoxyguanosine and comet assay in epirubicin-treated patients

Epirubicin fights cancer through topoisomerase II inhibition, hence producing DNA strand breaks that finally lead to cell apoptosis. But anthracyclines produce free radicals that may explain their adverse effects. Dexrazoxane--an iron chelator--was proven to decrease free radical production and anthracycline cardiotoxicity. In this article, we report the concentrations of cellular 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) relative to 2'-deoxyguanosine (dGuo), and comet assay results from a study including 20 cancer patients treated with epirubicin. Plasma concentrations of vitamins A, E, C and carotenoids are also reported. All data were obtained before and immediately after epirubicin infusion. The ratios of 8-Oxo-dGuo to dGuo were measured in leukocyte DNA by HPLC-coulometry after NaI extraction of nucleic acids. Vitamins A and E and carotenoids were measured by HPLC-spectrophotometry. Vitamin C was measured by HPLC-spectrofluorimetry. Median 8-oxo-dGuo/dGuo ratios increased significantly from 0.34 to 0.48 lesions per 100,000 bases while per cent of tail DNA increased from 3.47 to 3.94 after chemotherapy 8-Oxo-dGuo/dGuo and per cent of tail DNA medians remained in the normal range. Only vitamin C decreased significantly from 55.4 to 50.3 microM Decreases in vitamins A, E, lutein and zeaxanthin were not significant, but concentrations were below the lower limit of the normal range both before and after chemotherapy. Only the correlation between comet assay results and vitamin C concentrations was significant (rho =-0.517, p = 0.023). This study shows that cellular DNA is damaged by epirubicin-generated free radicals which produce the mutagenic modified base 8-oxo-dGuo and are responsible for strand breaks. However, strand breaks are created not only by free radicals but also by topoisomerase II inhibition. In a previous study we did not find any significant change in urinary 8-oxo-dGuo excretion after adriamycin treatment. However, 8-oxo-dGuo may have increased at the end of urine collection as DNA repair and subsequent kidney elimination are relatively slow processes. In another study, authors used GC-MS to detect 8-oxo-dGuo in DNA and did not find any change after prolonged adriamycin infusion. Reasons for these apparent discrepancies are discussed.     

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine as a biomarker in type 2 diabetes

The increasing prevalence of diabetes together with the associated morbidity and mortality calls for additional preventive and therapeutic strategies. New biomarkers that can be used in therapy control and risk stratification as alternatives to current methods are needed and can facilitate a more individualized and sufficient treatment of diabetes. Evidence derived from both epidemiological and mechanistic studies suggests that oxidative stress has an important role in mediating the pathologies of diabetic complications. A marker of intracellular oxidative stress that potentially could be used as a valuable biomarker in diabetes is the DNA oxidation marker 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), which can be assessed noninvasively in the urine, with minimal discomfort for the patient. In this review the analytical validity of 8-oxodG is addressed by highlighting important methodological issues. The available epidemiological evidence regarding urinary 8-oxodG and type 2 diabetes is presented. A possible role for DNA oxidation in cancer development in type 2 diabetes patients is discussed, followed by an evaluation of the potential of urinary 8-oxodG as a clinical biomarker in type 2 diabetes.     

Higher activity of 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) coincides with lower background levels of 8-oxo-2'-deoxyguanosine in DNA of fetal compared with maternal mouse organs.

Mammalian homologues of Escherichia coli MutT, a protein having 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) activity, are thought to play the same role in preventing the incorporation of promutagenic 8-oxo-2'-deoxyguanosine (8-oxo-dG) into DNA. One could thus expect that higher activity of 8-oxo-dGTPase should correlate with a lower background level of 8-oxo-dG in nuclear DNA. During transplacental carcinogenesis experiments, in control healthy Swiss mice on day 18 of gestation we found consistently lower levels of 8-oxo-dG in DNA in fetal livers and lungs (1.74+/-0.04 SE and 1.49+/-0.08 SE 8-oxo-dG/10(5) dG, respectively; pooled organs of fetuses of 8 dams) as compared with maternal organs (3.05+/-0.20 SE and 3.08+/-0.17 SE 8-oxo-dG/10(5) dG, respectively; n = 8). The 8-oxo-dGTPase activity determination in the same organs revealed that the lower levels of 8-oxo-dG in fetal DNA did, indeed, coincide with higher 8-oxo-dGTPase activity (48.8+/-2.6 SE and 52.5+/-2.5 SE U/mg protein in livers and lungs, respectively); and vice versa, higher 8-oxo-dG levels in DNA of maternal organs were associated with lower levels of 8-oxo-dGTPase activity (24.3+/-1.3 SE and 4.7+/-0.6 SE U/mg protein, as above). Without excluding other reasons for the relatively low 8-oxo-dG background in DNA of fetal tissues (e.g., higher level of antioxidants and antioxidative enzymes; more efficient DNA repair), this inverse relationship may support or at least does not contradict the concept of a guardian role of 8-oxo-dGTPase against 8-oxo-dGTP mutagenicity in mammalian cells.     

Harmonising measurements of 8-oxo-7,8-dihydro-2'-deoxyguanosine in cellular DNA and urine

Levels of oxidatively damaged cellular DNA and urinary excretion of damaged 2'-deoxyribonuclosides are widely measured in biomonitoring studies examining the role of oxidative stress induced by environmental exposures, lifestyle factors and development of disease. This has promoted efforts to harmonise measurements of oxidised guanine nucleobases by the variety of analytical approaches for DNA and urinary levels of damage, in multi-laboratory trials that are centred in Europe. The large inter-laboratory variation reported of values of oxidatively damaged DNA is reduced by harmonising assay protocols. Recent attention on optimal conditions for the comet assay may lead to better understanding of the most critical steps in procedure, which generate variation in DNA damage levels between laboratories. Measurements of urinary excretion of oxidatively generated 8-oxo-7,8-dihydro-2'-deoxyguanosine also show large differences between different methods, where chromatographic techniques generally show more reliable results than antibody-based methods. In this case, standardising calibrants is aimed at improving within technique agreement.     

Measurement of 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine in DNA and human urine by high performance liquid chromatography-electrospray tandem mass spectrometry

A method for the determination of 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine in DNA and urine by High Performance Liquid Chromatography (HPLC)-Tandem Mass Spectrometry is described. For the urine samples there is no sample preparation except for addition of buffer and internal standards followed by redissolvation of precipitate containing 8-oxo-2'-deoxyguanosine and a centrifugation step before the samples are injected onto the HPLC column. The detection limit for 8-oxo-2'-deoxyguanosine and 8-oxo-2'-deoxyadenosine is approximately 0.3 nM corresponding to 7.5 fmol injected. Long runs, that is, > 50 samples, can be analyzed with only minimal loss of sensitivity. The concentrations excreted into urine samples from humans are between 1 and 100 nM for 8-oxo-2'-deoxyguanosine and below 0.3 nM for 8-oxo-2'-deoxyadenosine. In calf thymus DNA levels down to about 1 oxidized guanosine and adenosine per 10(6) unmodified bases can be detected. High levels of 8-oxo-2'-deoxyguanosine were found, 30 per 10(6) 2'-deoxyguanosine, levels of 8-oxo-2'-deoxyadenosine are at or below the detection limit. These findings indicate that High Performance Liquid Chromatography-Tandem Mass Spectrometry is a highly sensitive and specific method for analysis of oxidative DNA modifications in tissue as well as for analysis of excretion of oxidized nucleotides into urine that ensures a minimum artifact formation.     

Evidence for attenuated cellular 8-oxo-7,8-dihydro-2'-deoxyguanosine removal in cancer patients

Measurement of the products of oxidatively damaged DNA in urine is a frequently used means by which oxidative stress may be assessed non-invasively. We believe that urinary DNA lesions, in addition to being biomarkers of oxidative stress, can potentially provide more specific information, for example, a reflection of repair activity. We used high-performance liquid chromatography prepurification, with gas chromatography-mass spectrometry (LC-GC-MS) and ELISA to the analysis of a number of oxidative [e.g., 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), 8-oxo-7,8-dihydro-guanine, 5-(hydroxymethyl)uracil], non-oxidative (cyclobutane thymine dimers) and oligomeric DNA products in urine. We analysed spot urine samples from 20 healthy subjects, and 20 age- and sex-matched cancer patients. Mononuclear cell DNA 8-oxodG levels were assessed by LC-EC. The data support our proposal that urinary DNA lesion products are predominantly derived from DNA repair. Furthermore, analysis of DNA and urinary 8-oxodG in cancer patients and controls suggested reduced repair activity towards this lesion marker in these patients.     

Comparison of results from different laboratories in measuring 8-oxo-2'-deoxyguanosine in synthetic oligonucleotides

The European Standards Committee on Oxidative DNA Damage (ESCODD) was set up in 1997 to resolve methodological problems and to reach agreement on the basal level of 8-oxo-2'-deoxyguanosine (8-oxodG) in biological samples. In the present ESCODD trial 6 samples of 8-oxodG-containing oligonucleotides with different ratios of 8-oxodG/2'-deoxyadenosine (dAdo) were sent to 25 laboratories throughout Europe. The methods used were HPLC with electrochemical detection (amperometric or coulometric), GC-MS or LC-MS-MS. The LC-MS-MS and the coulometric HPLC analyses gave 8-oxodG concentrations within 53 and 73% of expected values, respectively, whereas the amperometric HPLC and GC-MS consistently overestimated the 8-oxodG concentration by several fold. As the oligonucleotides contained no 2'-deoxyguanosine (dGuo), this was not due to artificial oxidation. On the contrary, in most cases the concentrations of dAdo and thymidine (dThd), used as estimates for non-oxidised DNA bases were underestimated, though a few laboratories overestimated the lowest concentration samples containing 8 and 20 μM, respectively. In one-third of the reported results, the ratio of 8-oxodG/10 5 dAdo was within 25% of the calculated value in the oligonucleotide samples and in half of the results the coefficient of variation in duplicate samples was less than 10%. The coefficients of variation were higher for the dAdo concentrations than for 8-oxodG. Our findings strongly indicate that careful quality control must be applied to the analytical procedures for 8-oxodG and very importantly also to the procedures for non-modified 2'-deoxyribonucleosides. We recommend the use of synthetic oligonucleotides for this purpose.     

Translesion synthesis of 7,8-dihydro-8-oxo-2'-deoxyguanosine by DNA polymerase eta in vivo

7,8-Dihydro-8-oxo-2′-deoxyguanosine (8-oxo-dG) is one of the most common DNA lesions induced by oxidative stress. This lesion can be bypassed by DNA polymerase eta (Pol η) using in vitro translesion synthesis (TLS) reactions. However, the role that Pol η plays in vivo contributing to 8-oxo-dG mutagenesis remains unclear. To clarify the role of Pol η in 8-oxo-dG mutagenesis, we have used an siRNA knockdown approach in combination with a supF shuttle vector (pSP189) which replicates in mammalian cells. The pSP189 plasmid was treated with methylene blue plus light (MBL), which produces predominantly 8-oxo-dG in DNA, and was then replicated in GM637 cells in presence of siRNA that knocks down the expression of Pol η, or in XP-V cells, which lack functional Pol η. The mutant frequencies were increased in the Pol η siRNA knockdown cells and in XP-V cells relative to control, meaning that Pol η plays an important role in preventing 8-oxo-dG mutagenesis. In the same system, knockdown of OGG1 also led to an increase in mutagenesis. Neither the type of mutations nor their distribution along the supF gene were significantly different between control and target specific siRNA-transfected cells (or XP-V cells) and were predominantly G to T transversions. These results show that Pol η has an important role in error-free 8-oxo-dG lesion bypass and avoidance of oxidative stress-induced mutagenesis in vivo.     

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), a reliable oxidative stress marker in hypertension

The potential use of oxidative stress products as disease markers and progression is an important aspect of biomedical research. In the present study, the quantification of urine 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) concentration has been used to express the oxidation status of hypertensive subjects.

8-oxo-dG has been simultaneously isolated and assayed in nuclear (nDNA) and mitochondrial DNA (mtDNA). In addition, oxidative stress of mononuclear cells has been estimated by means of GSH and GSSG levels and GSSG/GSH ratio in hypertensive subjects before and after antihypertensive treatment. It is shown that oxidative stress decreases significantly in hypertensive patients after treatment the effect being accompanied by reduction of their blood pressure.

A significant correlation is observed comparing the yield of urine 8-oxo-dG and that isolated from mitochondria DNA. Moreover, urinary excretion of 8-oxo-dG also correlates with the GSSG/GSH ratio of cells. Conclusion: urine 8-oxo-dG assay is a good marker for monitoring oxidative stress changes in hypertensives.     

Kinetics of phosphate-mediated oxidation of ferrous iron and formation of 8-oxo-2'-deoxyguanosine in solutions of free 2'-deoxyguanosine and calf thymus DNA

Formation of 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) in solutions of free 2'-deoxyguanosine (dG) and calf thymus DNA (DNA) was compared for the diffusion-dependent and localised production of oxygen radicals from phosphate-mediated oxidation of ferrous iron (Fe2+) to ferric iron (Fe3+). The oxidation of Fe2+ to Fe3+ was followed at 304 nm at pH 7.2 under aerobic conditions. Given that the concentration of Fe2+ >or=phosphate concentration, the rate of Fe2+ oxidation was significantly higher in DNA-phosphate as compared for the same concentration of inorganic phosphate. Phosphate catalysed oxidation of ferrous ions in solutions of dG or DNA led through the production of reactive oxygen species to the formation of 8-oxo-dG. The yield of 8-oxo-dG in solutions of dG or DNA correlated positively with the inorganic-/DNA-phosphate concentrations as well as with the concentrations of ferrous ions added. The yield of 8-oxo-dG per unit oxidised Fe2+ were similar for dG and DNA; thus, it differed markedly from radiation-induced 8-oxo-dG, where the yield in DNA was several fold higher.For DNA in solution, the localisation of the phosphate ferrous iron complex relative to the target is an important factor for the yield of 8-oxo-dG. This was supported from the observation that the yield of 8-oxo-dG in solutions of dG was significantly increased over that in DNA only when Fe2+ was oxidised in a high excess of inorganic phosphate (50 mM) and from the lower protection of DNA damage by the radical scavenger (hydroxymethyl)aminomethane (Tris)-HCl.     

Solid-phase synthesis of DNA fragments containing the modified base 7-hydro-8-oxo-2'-deoxyguanosine.

The 5'-(4,4'-dimethoxytrityl) protected 3'-(2-cyanoethoxy)-N,N-diisopropylphosphoramidite of 7-hydro-8-oxo-2'-deoxy-guanosine, the exocyclic amino and lactam functions of which are protected with acetyl and diphenylcarbamoyl groups, respectively, has been prepared from the 8-bromo derivatives of deoxy- and riboguanosine. This synthon, in combination with standard d-nucleoside 3'-(2-cyanoethoxy)-N,N-diisopropylphosphoramidites, was applied successfully to a solid-phase synthesis. Well-defined oligodeoxyribonucleotides containing a 7-hydro-8-oxo-2'-deoxyguanosine residue at predetermined positions were obtained after deprotection with methanolic ammonia and purification by gel filtration.     

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine and 5-(hydroxymethyl) uracil in smokers

Cigarette smoke is known to generate free radicals by various mechanisms. In this study involving 30 non-smokers and 30 smokers, we show that urinary excretion of 5-(hydroxymethyl) uracil (HMUra) was not different in the two groups (6.54±2.07 vs. 6.70±1.68 nmol/mmol creatinine). In contrast, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) excretion increased by 16% (1.16±0.35 vs. 1.35±0.50 nmol/mmol creatinine, p=0.039). Results concerning 8-oxo-dGuo are in agreement with those of previous studies. We observed significant multiple correlations between HMUra and creatinine (r_p=0.44), BMI (r_p=-0.27) and nicotine derivatives (r_p=0.26). Multiple correlation analysis showed relations between 8-oxo-dGuo on the one hand, and: creatinine (r_p=0.36), nicotine derivatives (r_p=0.29), BMI (r_p=-0.24) on the other.     

Chromatographic determination of 8-oxo-7,8-dihydro-2'-deoxyguanosine in cellular DNA: a validation study

Although a series of biomarkers are widely used for the estimation of oxidative damage to biomolecules, validations of the analytical methods have seldom been presented. Formal validation, that is the study of the analytical performances of a method, is however recognized as the best safeguard against the generation and publication of data with low reliability. Classical validation parameters were investigated for the determination of an oxidative stress biomarker, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) in cellular DNA, by high-performance liquid chromatography coupled to amperometric detection (HPLC-EC); this modified base is increasingly considered as a marker of oxidative damage to DNA, but many questions are still raised on the analytical methods in use. Upon a rigorous statistical evaluation of the quality criteria currently required for assays in biological media, including selectivity, linearity, accuracy, repeatability, sensitivity, limits of detection and quantification, ruggedness and storage at different stop points in the procedure, the HPLC-EC assay method is found mostly reliable. The present validation attempt demonstrates that (i) the HPLC-EC assay of 8-oxo-dG provides consistent data allowing to reliably detect an increase of this biomarker in cellular DNA; (ii) a harsh oxidative stress does not hinder the enzymatic digestion of DNA by nuclease P1; and (iii) the analytical results must be expressed relative to the internal standard dG which significantly improves both repeatability and sensitivity. Whereas the described assay minimizes the artifactual production of the analyte from processing and storage, this cannot be totally ruled out; the true 8-oxo-dG base levels still lack a definitive assay method, which remains a considerable analytical challenge and the object of controversy.     

Inhibition of 8-oxo-2'-deoxyguanosine 5'-triphosphate pyrophosphohydrolase (8-oxo-dGTPase) activity of the antimutagenic human MTH1 protein by nucleoside 5'-diphosphates

The hMTH1 protein, a human homologue of E. coli MutT protein, is an enzyme converting 8-oxo-2'-deoxyguanosine 5'-triphosphate (8-oxo-dGTP) to 8-oxo-2'-deoxyguanosine 5'-monophosphate (8-oxo-dGMP) and inorganic pyrophosphate. It is thought to play an antimutagenic role by preventing the incorporation of promutagenic 8-oxo-dGTP into DNA. As found in our previous investigations, 8-oxo-2'-deoxyguanosine 5'-diphosphate (8-oxo-dGDP) strongly inhibited 8-oxo-dGTPase activity of MTH1. Following this finding, in the present study we have tested the canonical ribo- and deoxyribonucleoside 5'-diphosphates (NDPs and dNDPs) for possible inhibition of 8-oxo-dGTP hydrolysis by hMTH1 extracted from CCRF-CEM cells (a human leukemia cell line). Among them, the strongest inhibitors appeared to be dGDP (Ki=74 microM), dADP (Ki=147 microM), and GDP (Ki=502 microM). Other dNDPs and NDPs, such as dCDP, dTDP, ADP, CDP, and UDP were much weaker inhibitors, with Ki in the millimolar range. Based on the present results and published data, we estimate that the strongest inhibitors, dGDP and dADP, at physiological concentrations not exceeding 5 microM and GDP at mean concentration of 30 microM, taken together, can decrease the cellular hMTH1 enzymatic activity vs. 8-oxo-dGTP (expected to remain below 500 pM) by up to 15%. The other five NDPs and dNDPs tested cannot markedly affect this activity.