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.     

Oxidised guanidinohydantoin (Ghox) and spiroiminodihydantoin (Sp) are major products of iron- and copper-mediated 8-oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine oxidation

8-Oxo-7,8-dihydroguanine (8-oxoGua), an important biomarker of DNA damage in oxidatively generated stress, is highly reactive towards further oxidation. Much work has been carried out to investigate the oxidation products of 8-oxoGua by one-electron oxidants, singlet oxygen, and peroxynitrite. This report details for the first time, the iron- and copper-mediated Fenton oxidation of 8-oxoGua and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). Oxidised guanidinohydantoin (Gh(ox)) was detected as the major product of oxidation of 8-oxoGua with iron or copper and hydrogen peroxide, both at pH 7 and pH 11. Oxaluric acid was identified as a final product of 8-oxoGua oxidation. 8-oxodGuo was subjected to oxidation under the same conditions as 8-oxoGua. However, dGh(ox) was not generated. Instead, spiroiminodihydantoin (Sp) was detected as the major product for both iron and copper mediated oxidation at pH 7. It was proposed that the oxidation of 8-oxoGua was initiated by its one-electron oxidation by the metal species, which leads to the reactive intermediate 8-oxoGua (+), which readily undergoes further oxidation. The product of 8-oxoGua and 8-oxodGuo oxidation was determined by the 2'-deoxyribose moiety of the 8-oxodGuo, not whether copper or iron was the metal involved in the oxidation.     

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.     

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 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.     

Oxidatively induced DNA-protein cross-linking between single-stranded binding protein and oligodeoxynucleotides containing 8-oxo-7,8-dihydro-2'-deoxyguanosine

The formation of covalent cross-links between amino acid side chains and DNA bases in DNA-protein complexes is a significant pathway in oxidative damage to the genome, yet much remains to be learned about their chemical structures and mechanisms of formation. In the present study, DNA-protein cross-links (DPCs) were formed between synthetic oligodeoxynucleotides containing an 8-oxo-7,8-dihydro-2'deoxyguanosine (OG) or an 8-oxo-7,8-dihydro-2'-deoxyadenosine (OA) nucleotide and Escherichia coli singled-stranded binding protein (SSB) under oxidative conditions. Studies with various sequences indicated that DNA homopolymers and those lacking 8-oxopurines were less reactive toward DPC formation. DPCs were formed in the presence of HOCl, peroxynitrite, and the one-electron oxidants Na(2)IrCl(6), Na(2)IrBr(6), and Na(3)Fe(CN)(6). Protein-protein cross-linking was also observed, particularly for oxidants of high reduction potential such as Na(2)IrCl(6). The adducted oligodeoxynucleotides were sensitive to hot piperidine treatment leading to strand scission at the site of cross-linking. In addition, the covalent cross-links were somewhat heat and acid labile, which may be related to the difficulties encountered in obtaining complete characterization of trypsin digests of the DPCs. However, model reactions involving the single amino acids lysine, arginine, and tyrosine, residues known to be involved in base contacts in the DNA:SSB complex, could be studied, and the adduct formed between N(alpha)-acetyllysine methyl ester and an 18-mer containing OG was tentatively characterized by electrospray ionization mass spectrometry as analogues of spiroiminodihydantoin and guanidinohydantoin. A mechanism involving nucleophilic attack of an amino acid side chain (e.g. the epsilon-amino group of lysine) at C5 of a 2-electron oxidized form of OG is proposed.     

8-Oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanosine levels in human urine do not depend on diet

In the present study, we used the method involving HPLC pre-purification followed by gas chromatography with isotope dilution mass spectrometric detection for the determination of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) and 8-oxo-7,8-dihydroguanine (8-oxoGua) in human urine. The mean levels of 8-oxoGua and 8-oxodGuo in the urine samples of the subjects on unrestricted diet were respectively 1.87 nmol/kg 24 h (±0.90) and 0.83 nmol/kg 24h (±0.49), and in the case of the groups studied, they did not depend on the applied diet. The sum of the amounts of both compounds in urine can give information about the formation rate of 8-oxoGua in cellular DNA. It is also likely that the levels of modified nucleo-base/side in urine sample are reflective of the involvement of different repair pathways responsible for the removal of 8-oxodGuo from DNA, namely base excision repair (BER) and nucleotide excision repair (NER).     

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.     

Effects of arsenic exposure among semiconductor workers: a cautionary note on urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine

Arsenic is a notorious environmental toxicant and was found to cause oxidative stress in cultured cells and animals. However, little work has been done in human studies, especially for the population occupationally exposed to arsenic. In order to investigate the effect of occupational exposure to arsenic in oxidative stress, we measured urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) from 90 semiconductor workers including 50 exposed and 40 nonexposed subjects. A highly sensitive and specific isotope dilution LC-MS/MS method was used for quantification of 8-oxodGuo. The levels of inorganic arsenic (iAs3+, iAs5+), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) in urine were determined by high-performance liquid chromatography-flow injection atomic absorption spectrometry (HPLC-FIAAS). Results showed that the mean urinary concentrations of total arsenic and 8-oxodGuo were significantly higher for exposed workers compared with the nonexposed workers. In addition, elevated urinary 8-oxodGuo concentrations of exposed workers were correlated with urinary levels of MMA (r = 0.44, P < 0.005) and the extent of primary methylation (the ratio of MMA to inorganic arsenic) (r = 0.40, P < 0.005). These findings suggested that occupational exposure to arsenic could result in the induction of oxidative stress. The presence and/or formation of MMA could play an important role in arsenic-involved injuries.     

Influence of flavan-3-ols and procyanidins on UVC-mediated formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine in isolated DNA

The flavan-3-ols (-)-epicatechin (epicatechin) and (+)-catechin (catechin) and their related oligomers (procyanidins) isolated from cocoa were assayed for their capacity to inhibit the UVC-mediated formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxo(8)dG) in calf thymus DNA. The above-mentioned compounds inhibited oxo(8)dG production in a concentration- and time-dependent manner. After 30 min of irradiation (30 kJ/m(2)), 0.1, 1.0, 10, and 100 microM epicatechin inhibited oxo(8)dG formation by 20, 36, 64, and 74%, respectively. For the same dose of UVC, 0.1, 1.0, 10, and 100 microM catechin inhibited oxo(8)dG formation by 1, 23, 50, and 70%, respectively. Epicatechin was more efficient than catechin with respect to inhibiting oxo(8)dG formation (IC(50) 1.7 +/- 0.7 vs 4.0 +/- 0.7 microM). Monomer, tetramer, and hexamer fractions were equally effective in inhibiting oxo(8)dG formation when assayed at 10 microM monomer equivalent concentration. At similar concentrations (1-50 microM), the inhibition of the UVC-mediated oxo(8)dG formation by flavan-3-ols and procyanidins was in the range of that of alpha-tocopherol, Trolox, ascorbate, and glutathione. These results support the concept that flavan-3-ols and their related procyanidins can protect DNA from oxidation at concentrations that can be physiologically relevant. Both epimerism and degree of oligomerization are important determinants of the antioxidant activity of flavan-3-ols and procyanidins.     

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.     

Mutagenicity of secondary oxidation products of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-hydroxy-2'- deoxyguanosine 5'-triphosphate)

8-Oxo-7,8-dihydroguanine (8-hydroxyguanine) is oxidized more easily than normal nucleobases, which can produce spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). These secondary oxidation products of 8-oxo-7,8-dihydroguanine are highly mutagenic when formed within DNA. To evaluate the mutagenicity of the corresponding oxidation products of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-hydroxy-2'- deoxyguanosine 5'-triphosphate) in the nucleotide pool, Escherichia coli cells deficient in the mutT gene were treated with H(2)O(2), and the induced mutations were analyzed. Moreover, the 2'-deoxyriboside 5'-triphosphate derivatives of Sp and Gh were also introduced into competent E. coli cells. The H(2)O(2) treatment of mutT E. coli cells resulted in increase of G:C → T:A and A:T → T:A mutations. However, the incorporation of exogenous Sp and Gh 2'-deoxyribonucleotides did not significantly increase the mutation frequency. These results suggested that the oxidation product(s) of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate induces G:C → T:A and A:T → T:A mutations, and that the 2'-deoxyriboside 5'-triphosphate derivatives of Sp and Gh exhibit quite weak mutagenicity, in contrast to the bases in DNA.     

Renal excretion of 8-oxo-7,8-dihydro-2'-deoxyguanosine in Wistar rats with increased O(2) consumption due to cold stress

DNA damage by reactive oxygen species is of special interest in the development of cancer and in aging. The renally excreted amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (oxo(8)dG) is a potential noninvasive marker of oxidative DNA damage. The respiratory chain of mitochondria is one source for the formation of reactive oxygen species. In the present study we investigated in Wistar rats (n = 7; mean body weight at start, 307.4 +/- 11 g) the effect of an increased O(2) consumption, i.e., energy expenditure, due to cold stress on the renally excreted amount of oxo(8)dG. First, the rats were housed for 4 days at 23.5 degrees C (basic period, BP), and then for 6 days at 10 degrees C (cold stress period, CSP), and finally for 3 days at 23.5 degrees C (recovery period, RP). The O(2) consumption (L O(2)/day/kg weight) was significantly (P < 0.0001) on average 50% higher in CSP (69.0 +/- 3.9) than in BP (45.8 +/- 4.8), and similar in BP and RP (44.3 +/- 5.4). The average renal excretion of oxo(8)dG (pmol/day/kg weight) was significantly (P < 0.025) on average 13% higher in CSP (375.5 +/- 27.7) than in BP (333.2 +/- 47. 4) and similar in BP and RP (331.8 +/- 34.3). Maximum increase in oxo(8)dG excretion of on average 17% was on the third to fifth day of the CSP. This study reveals that an increase in O(2) consumption of 50% resulted in a much lower increase in the renal excretion of oxo(8)dG.     

A highly conserved Tyrosine residue of family B DNA polymerases contributes to dictate translesion synthesis past 8-oxo-7,8-dihydro-2'-deoxyguanosine

The harmfulness of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8oxodG) damage resides on its dual coding potential, as it can pair with the correct dCMP (dC) or the incorrect dAMP (dA). Here, we investigate the translesional synthesis ability of family B ϕ29 DNA polymerase on 8oxodG-containing templates. We show that this polymerase preferentially inserts dC opposite 8oxodG, its 3′–5′ exonuclease activity acting indistinctly on both dA or dC primer terminus. In addition, ϕ29 DNA polymerase shows a favoured extension of the 8oxodG/dA pair, but with an efficiency much lower than that of the canonical dG/dC pair. Additionally, we have analysed the role of the invariant tyrosine from motif B of family B DNA polymerases in translesional synthesis past 8oxodG, replacing the corresponding ϕ29 DNA polymerase Tyr390 by Phe or Ser. The lack of the aromatic portion in mutant Y390S led to a lost of discrimination against dA insertion opposite 8oxodG. On the contrary, the absence of the hydroxyl group in the Y390F mutant precluded the favoured extension of 8oxodG:dA base pair with respect to 8oxodG:dC. Based on the results obtained, we propose that this Tyr residue contributes to dictate nucleotide insertion and extension preferences during translesion synthesis past 8oxodG by family B replicases.     

The effect of the 2-amino group of 7,8-dihydro-8-oxo-2'-deoxyguanosine on translesion synthesis and duplex stability

Replication of DNA containing 7,8-dihydro-8-oxo-2′-deoxyguanosine (OxodG) gives rise to G → T transversions. The syn-isomer of the lesion directs misincorporation of 2′-deoxyadenosine (dA) opposite it. We investigated the role of the 2-amino substituent on duplex thermal stability and in replication using 7,8-dihydro-8-oxo-2′-deoxyinosine (OxodI). Oligonucleotides containing OxodI at defined sites were chemically synthesized via solid phase synthesis. Translesion incorporation opposite OxodI was compared with 7,8-dihydro-8-oxo-2′-deoxyguanosine (OxodG), 2′-deoxyinosine (dI) and 2′-deoxyguanosine (dG) in otherwise identical templates. The Klenow exo⁻ fragment of Escherichia coli DNA polymerase I incorporated 2′-deoxyadenosine (dA) six times more frequently than 2′-deoxycytidine (dC) opposite OxodI. Preferential translesion incorporation of dA was unique to OxodI. UV-melting experiments revealed that DNA containing OxodI opposite dA is more stable than when the modified nucleotide is opposed by dC. These data suggest that while duplex DNA accommodates the 2-amino group in syn-OxodG, this substituent is thermally destabilizing and does not provide a kinetic inducement for replication by Klenow exo⁻.     

Promotion of radiation-induced formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine by nitro 5-deazaflavin derivatives.

6-Nitro- and 8-nitro-5-deazaflavin derivatives have been found to enhance prominently the radiation-induced formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) at the expense of formation of 2,6-diamino-4-hydroxy-5-formamidopyrimidine nucleosides (FapydGuo) both in deaerated and in N(2)O saturated aqueous 2'-deoxyguanosine solutions. The radiosensitizing capacity of a 9-nitro-5-deazflavin derivative was observed only in the N(2)O saturated aqueous solutions.     

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.     

In isolated DNA, formamidopyrimidine-DNA glycosylase-sensitive sites determined by electrophoresis correspond to the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine by HPLC-ECD.

Several methods have been developed for determining the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in DNA. In the present study, we compared an electrophoretic method that uses formamidopyrimidine-DNA glycosylase (FPG protein) with a HPLC-ECD method. Firstly, we produced 8-oxodG in lambda DNA with methylene blue and visible light and cleaved it in one-half of the modified DNA enzymatically with FPG protein. Then, we determined the number of FPG protein-sensitive sites by electrophoresis (Y) and the number of 8-oxodGs by HPLC-ECD (X) per 10(5)dG of isolated DNA. Simple regression analysis of the data showed Y=1.07X+1.52 to be the most likely relationship. The correlation coefficient was 0.97. The values obtained by the two methods were very similar. This result is noteworthy because the number of FPG protein-sensitive sites determined by other methods have not yet come close to the number obtained by HPLC-ECD. Thus, this method might be more quantitative than other methods that measure FPG protein-sensitive sites. Another reason this electrophoresis method might be more useful than HPLC-ECD is that we can determine some other types of oxidative DNA damage well, by changing the DNA glycosylase.