A DNA aptamer sensor for 8-oxo-7,8-dihydroguanine

Abasic site-containing DNA duplex is a versatile structural motif that can be used for the design of purine aptamers and sensors. In this study, several modifications were introduced to the abasic site to explore possible specific binding of free 8-oxoG, a product of DNA base excision repair. The nucleoside opposite the abasic site was replaced by pyrrolo-dC as a reporter group. Binding of 8-oxoG quenched pyrrolo-dC fluorescence by as much as 70%. In contrast, adenine, guanine, thymine, and cytosine showed only minimal fluorescence quenching effect. The best aptamer binds 8-oxoG with a dissociation constant of 5.5±0.8μM. This sensor can be used to accurately measure 8-oxoG concentrations in the presence of guanine.     

DNA repair of 8-oxo-7,8-dihydroguanine lesions in Porphyromonas gingivalis

The persistence of Porphyromonas gingivalis in the inflammatory environment of the periodontal pocket requires an ability to overcome oxidative stress. DNA damage is a major consequence of oxidative stress. Unlike the case for other organisms, our previous report suggests a role for a non-base excision repair mechanism for the removal of 8-oxo-7,8-dihydroguanine (8-oxo-G) in P. gingivalis. Because the uvrB gene is known to be important in nucleotide excision repair, the role of this gene in the repair of oxidative stress-induced DNA damage was investigated. A 3.1-kb fragment containing the uvrB gene was PCR amplified from the chromosomal DNA of P. gingivalis W83. This gene was insertionally inactivated using the ermF-ermAM antibiotic cassette and used to create a uvrB-deficient mutant by allelic exchange. When plated on brucella blood agar, the mutant strain, designated P. gingivalis FLL144, was similar in black pigmentation and beta-hemolysis to the parent strain. In addition, P. gingivalis FLL144 demonstrated no significant difference in growth rate, proteolytic activity, or sensitivity to hydrogen peroxide from that of the parent strain. However, in contrast to the wild type, P. gingivalis FLL144 was significantly sensitive to UV irradiation. The enzymatic removal of 8-oxo-G from duplex DNA was unaffected by the inactivation of the uvrB gene. DNA affinity fractionation identified unique proteins that preferentially bound to the oligonucleotide fragment carrying the 8-oxo-G lesion. Collectively, these results suggest that the repair of oxidative stress-induced DNA damage involving 8-oxo-G may occur by a still undescribed mechanism in P. gingivalis.     

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.     

Urinary excretion of 8-oxo-7,8-dihydroguanine as biomarker of oxidative damage to DNA

Oxidatively damaged DNA may be important in carcinogenesis. 8-Oxo-7,8-dihydroguanine (8-oxoGua) is an abundant and mutagenic lesion excised by oxoguanine DNA glycosylase 1 (OGG1) and measurable in urine or plasma by chromatographic methods with electrochemical or mass spectrometric detectors, reflecting the rate of damage in steady state. A common genetic OGG1 variant may affect the activity and was associated with increased levels of oxidized purines in leukocytes without apparent effect on 8-oxoGua excretion or major change in cancer risk. 8-OxoGua excretion has been associated with exposure to air pollution, toxic metals, tobacco smoke and low plasma antioxidant levels, whereas fruit and vegetable intake or dietary interventions showed no association. In rodent studies some types of feed may be source of 8-oxoGua in collected urine. Of cancer therapies, cisplatin increased 8-oxoGua excretion, whereas radiotherapy only showed such effects in experimental animals. Case-control studies found high excretion of 8-oxoGua in relation to cancer, dementia and celiac disease but not hemochromatosis, although associations could be a consequence rather than reflecting causality of disease. One prospective study found increased risk of developing lung cancer among non-smokers associated with high excretion of 8-oxoGua. Urinary excretion of 8-oxoGua is a promising biomarker of oxidatively damaged DNA.     

The oxidation of 8-oxo-7,8-dihydroguanine by iodine

8-Oxo-7,8-dihydroguanine was specifically oxidized by iodine with aqueous KI. Under acidic conditions, the major product was dehydro-guanidinohydantoin. Under basic conditions, two diastereoisomers of spirohydantoin were chiefly obtained. In addition, unstable diimine was detected for the first time.     

Enhanced mutagenic potential of 8-oxo-7,8-dihydroguanine when present within a clustered DNA damage site

The formation of clustered DNA damage sites is a unique feature of ionizing radiation. Recent studies have shown that the repair of lesions within clusters may be compromised, but little is understood about the mutagenic consequences of such damage sites. Using a plasmid-based method, damaged DNA containing uracil positioned at 1–5 bp separations from 8-oxo-7,8-dihydroguanine on the complementary strand was transfected into wild-type Escherichia coli or into strains lacking the DNA glycosylases Fpg and MutY. Mutation frequencies were found to be significantly higher for clustered damage sites than for single lesions. The loss of MutY gave a large relative increase in mutation frequency and a strain lacking both Fpg and MutY showed even higher mutation frequencies, up to nearly 40% of rescued plasmid. In these strains, the mutation frequency decreases with increasing spacing of the uracil from the 8-oxo-7,8-dihydroguanine site. Sequencing of plasmid DNA carrying clustered damage, following rescue from bacteria, showed that almost all of the mutations are GC→TA transversions. The data suggest that at clustered damage sites, depending on lesion spacing, the action of Fpg is compromised and post-replication processing of lesions by MutY is the most important mechanism for protection against mutagenesis.     

Larger yield of cyclobutane dimers than 8-oxo-7,8-dihydroguanine in the DNA of UVA-irradiated human skin cells

Exposure to solar ultraviolet light is the major cause of most skin cancers. While the genotoxic properties of UVB radiation are now well understood, the DNA damaging processes triggered by less energetic but more abundant UVA photons remain to be elucidated. Evidence has been provided for the induction of oxidative lesions to cellular DNA including strand breaks and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo). Formation of cyclobutane pyrimidine dimers (CPDs) has also been reported, mostly in rodent cells. In order to gain insights into the relevance of the latter photoproducts in UVA-mutagenesis of human skin, we quantified the level of 8-oxodGuo and CPDs within primary cultures of normal fibroblasts and keratinocytes using specific chromatographic assays. The yield of formation of CPDs was found to be higher than that of 8-oxodGuo in both cell types. In addition, CPDs were mostly TT derivatives, and neither (6-4) photoproducts nor Dewar valence isomers were detected. These observations are reminiscent of results obtained in rodent cells and suggest that a photosensitized triplet energy transfer occurs and that this reaction is more efficient than photooxidation of DNA components. The predominant formation of CPDs with respect to oxidative damage within normal human skin cells exposed to UVA radiation should be taken into account in photoprotection strategies.     

Biochemical identification of a hydroperoxide derivative of the free 8-oxo-7,8-dihydroguanine base

8-Oxo-7,8-dihydroguanine is one the most abundant base lesions in pro- and eukaryotic DNA. In mammalian cells, it is excised by the 8-oxoguanine DNA glycosylase (OGG1) during DNA base-excision repair, and the generated free 8-oxoG base is one of the DNA-derived biomarkers of oxidative stress in biological samples. The modification of 8-oxoG in the context of nucleoside and DNA has been the subject of many studies; however, the oxidative transformation of the free 8-oxoG base has not been described. By using biochemical and cell biological assays, we show that in the presence of molecular oxygen, the free 8-oxoG base transforms to a highly reactive hydroperoxide (8-oxoG*). Specifically, 8-oxoG* oxidizes Amplex red to resorufin, H(2)DCF to DCF, Fe(2+) to Fe(3+), and GSH to GSSG. This property of 8-oxoG* was diminished by treatment with catalase and glutathione peroxidase, but not superoxide dismutase. 8-OxoG* formation was prevented by reducing agents or nitrogen atmosphere. Its addition to CM-H(2)DCF-DA-loaded cells rapidly increased intracellular DCF fluorescence. There were no such properties observed for 8-oxodeoxyguanosine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, 2'-deoxyguanosine, guanine, adenine, guanosine, and 8-hydroxyadenine. These data imply that a free 8-oxoG base is more susceptible to oxidation than is its nucleoside form and, consequently, it stands as unique among intact and oxidatively modified purines.     

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).     

Incidence and persistence of 8-oxo-7,8-dihydroguanine within a hairpin intermediate exacerbates a toxic oxidation cycle associated with trinucleotide repeat expansion

The repair protein 8-oxo-7,8-dihydroguanine glycosylase (OGG1) initiates base excision repair (BER) in mammalian cells by removing the oxidized base 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Interestingly, OGG1 has been implicated in somatic expansion of the trinucleotide repeat (TNR) sequence CAG/CTG. Furthermore, a 'toxic oxidation cycle' has been proposed for age-dependent expansion in somatic cells. In this cycle, duplex TNR DNA is (1) oxidized by endogenous species; (2) BER is initiated by OGG1 and the DNA is further processed by AP endonuclease 1 (APE1); (3) a stem-loop hairpin forms during strand-displacement synthesis by polymerase β (pol β); (4) the hairpin is ligated and (5) incorporated into duplex DNA to generate an expanded CAG/CTG region. This expanded region is again subject to oxidation and the cycle continues. We reported previously that the hairpin adopted by TNR repeats contains a hot spot for oxidation. This finding prompted us to examine the possibility that the generation of a hairpin during a BER event exacerbates the toxic oxidation cycle due to accumulation of damage. Therefore, in this work we used mixed-sequence and TNR substrates containing a site-specific 8-oxoG lesion to define the kinetic parameters of human OGG1 (hOGG1) activity on duplex and hairpin substrates. We report that hOGG1 activity on TNR duplexes is indistinguishable from a mixed-sequence control. Thus, BER is initiated on TNR sequences as readily as non-repetitive DNA in order to start the toxic oxidation cycle. However, we find that for hairpin substrates hOGG1 has reduced affinity and excises 8-oxoG at a significantly slower rate as compared to duplexes. Therefore, 8-oxoG is expected to accumulate in the hairpin intermediate. This damage-containing hairpin can then be incorporated into duplex, resulting in an expanded TNR tract that now contains an oxidative lesion. Thus, the cycle restarts and the DNA can incrementally expand.     

Fidelity of nucleotide insertion at 8-oxo-7,8-dihydroguanine by mammalian DNA polymerase delta. Steady-state and pre-steady-state kinetic analysis

Nucleotide insertion opposite 8-oxo-7,8-dihydroguanine (8-oxoG) by fetal calf thymus DNA polymerase delta (pol delta) was examined by steady-state and pre-steady-state rapid quench kinetic analyses. In steady-state reactions with the accessory protein proliferating cell nuclear antigen (PCNA), pol delta preferred to incorporate dCTP opposite 8-oxoG with an efficiency of incorporation an order of magnitude lower than incorporation into unmodified DNA (mainly due to an increased K(m)). Pre-steady-state kinetic analysis of incorporation opposite 8-oxoG showed biphasic kinetics for incorporation of either dCTP or dATP, with rates similar to dCTP incorporation opposite G, large phosphorothioate effects (>100), and oligonucleotide dissociation apparently rate-limiting in the steady-state. Although pol delta preferred to incorporate dCTP (14% misincorporation of dATP) the extension past the A:8-oxoG mispair predominated. The presence of PCNA was found to be a more essential factor for nucleotide incorporation opposite 8-oxoG adducts than unmodified DNA, increased pre-steady-state rates of nucleotide incorporation by >2 orders of magnitude, and was essential for nucleotide extension beyond 8-oxoG. pol delta replication fidelity at 8-oxoG depends upon contributions from K(m), K(d)(dNTP), and rates of phosphodiester bond formation, and PCNA is an important accessory protein for incorporation and extension at 8-oxoG adducts.     

Association between 8-oxo-7,8-dihydroguanine excretion and risk of lung cancer in a prospective study

Oxidative damage to guanine (8-oxoGua) is one of the most abundant lesions induced by oxidative stress and documented mutagenic. 8-Oxoguanine DNA glycosylase 1 (OGG1) removes 8-oxoGua from DNA by excision. The urinary excretion of 8-oxoGua is a biomarker of exposure, reflecting the rate of damage in the steady state. The aim of this study was to investigate urinary 8-oxoGua as a risk factor for lung cancer. In a nested case-cohort design we examined associations between urinary excretion of 8-oxoGua and risk of lung cancer as well as potential interaction with the OGG1 Ser326Cys polymorphism in a population-based cohort of 25,717 men and 27,972 women aged 50-64 years with 3-7 years follow-up. We included 260 cases with lung cancer and a subcohort of 263 individuals matched on sex, age, and smoking duration for comparison. Urine collected at entry was analysed for 8-oxoGua by HPLC with electrochemical detection. There was no significant effect of smoking or OGG1 genotype on the excretion of 8-oxoGua. Overall the incidence rate ratio (IRR) (95% confidence interval) of lung cancer was 1.06 (0.97-1.15) per doubling of 8-oxoGua excretion. The association between lung cancer risk and 8-oxoGua excretion was significant among men [IRR: 1.17 (1.03-1.31)], never-smokers [IRR: 9.94 (1.04-94.7)], and former smokers [IRR: 1.19 (1.07-1.33)]. There was no significant interaction with the OGG1 genotype, although the IRR was 1.14 (0.98-1.34) among subjects homozygous for Cys326. The association between urinary 8-oxoGua excretion and lung cancer risk among former and never-smokers suggests that oxidative stress with damage to DNA is important in this group.     

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.     

Substantial decrease of urinary 8-oxo-7,8-dihydroguanine, a product of the base excision repair pathway, in DNA glycosylase defective mice

Genome integrity is maintained via removal (repair) of DNA lesions and an increased load of such DNA damage has been linked to numerous pathological conditions, including carcinogenesis and ageing. 8-Oxo-7,8-dihydroguanine is one of the most critical lesions of this type. The free 8-oxo-7,8-dihydroguanine produced by the action of a specific DNA glycosylase is a potential source of this compound in urine. To date, there has been no direct, experimental evidence demonstrating that urinary 8-oxo-7,8-dihydroguanine is produced by the base excision repair pathway. For clarification of this issue, we applied a recently developed methodology which involved high performance liquid chromatography pre-purification followed by gas chromatography with isotope dilution mass spectrometric detection to compare the urinary excretion rate of 8-oxo-7,8-dihydroguanine in wild type and OGG1 glycosylase knock out mice. Our study revealed a 26% reduction in urinary level of 8-oxo-7,8-dihydroguanine in OGG1 deficient mice in comparison with the wild type strain. This clearly indicates that the mouse OGG1 glycosylase contributes significantly to the generation of urinary 8-oxo-7,8-dihydroguanine. Therefore, urinary measurements of 8-oxo-7,8-dihydroguanine may be attributed to DNA damage and repair, which in turn suggests that they may be useful in studying associations between DNA repair and disease.     

8-oxo-7,8-dihydroguanine is removed by a nucleotide excision repair-like mechanism in Porphyromonas gingivalis W83

A consequence of oxidative stress is DNA damage. The survival of Porphyromonas gingivalis in the inflammatory microenvironment of the periodontal pocket requires an ability to overcome oxidative stress caused by reactive oxygen species (ROS). 8-oxo-7,8-dihydroguanine (8-oxoG) is typical of oxidative damage induced by ROS. There is no information on the presence of 8-oxoG in P. gingivalis under oxidative stress conditions or on a putative mechanism for its repair. High-pressure liquid chromatography with electrochemical detection analysis of chromosomal DNA revealed higher levels of 8-oxoG in P. gingivalis FLL92, a nonpigmented isogenic mutant, than in the wild-type strain. 8-oxoG repair activity was also increased in cell extracts from P. gingivalis FLL92 compared to those from the parent strain. Enzymatic removal of 8-oxoG was catalyzed by a nucleotide excision repair (NER)-like mechanism rather than the base excision repair (BER) observed in Escherichia coli. In addition, in comparison with other anaerobic periodontal pathogens, the removal of 8-oxoG was unique to P. gingivalis. Taken together, the increased 8-oxoG levels in P. gingivalis FLL92 could further support a role for the hemin layer as a unique mechanism in oxidative stress resistance in this organism. In addition, this is the first observation of an NER-like mechanism as the major mechanism for removal of 8-oxoG in P. gingivalis.     

Effects of base excision repair proteins on mutagenesis by 8-oxo-7,8-dihydroguanine (8-hydroxyguanine) paired with cytosine and adenine

8-Oxo-7,8-dihydroguanine (8-oxo-Gua, also known as 8-hydroxyguanine) is a major base lesion that is generated by reactive oxygen species in both the DNA and nucleotide pool. The role of DNA glycosylases, which initiate base excision repair, in the mutagenic processes of 8-oxo-Gua in DNA and 8-oxo-7,8-dihydro-2′-deoxyguanosine 5′-triphosphate (8-oxo-dGTP, also known as 8-hydroxy-2′-deoxyguanosine 5′-triphosphate) were investigated using supF shuttle plasmids propagated in human cells. The DNA glycosylases, OGG1, MUTYH, NTH1, and NEIL1, in 293T cells were individually knocked-down by siRNAs and plasmid DNAs containing an 8-oxo-Gua:C/8-oxo-Gua:A pair, and 8-oxo-dGTP plus unmodified plasmid DNA were then introduced into the knocked-down cells. The knock-down of OGG1, MUTYH, NTH1, and NEIL1 resulted in a significant increase in G:C → T:A transversions caused by the 8-oxo-Gua:C pair in the shuttle plasmid. The knock-down of MUTYH resulted in a reduction in A:T → C:G transversions induced by 8-oxo-dGTP and the 8-oxo-Gua:A pair, but the knockdown of OGG1, NTH1, and NEIL1 had no effect on mutagenesis. These results indicate that all of the above DNA glycosylases suppress mutations caused by 8-oxo-Gua:C in DNA. In contrast, it appears that MUTYH enhances A:T → C:G mutations caused by 8-oxo-dGTP.     

Unique response of double-stranded oligonucleotides containing a single 8-oxo-7,8-dihydroguanine to gamma rays in the frozen aqueous state at 77 K

Two kinds of double-stranded oligonucleotides containing a single 8-oxo-7,8-dihydroguanine were labeled with (32)P at their 5' ends and exposed to gamma rays in the frozen aqueous state at 77 K, where both direct and quasi-direct effects of ionizing radiation predominate. Analysis of the oligonucleotides with 20% denaturing polyacrylamide gel electrophoresis revealed no difference in the immediate induction of strand breaks between oligonucleotides containing 8-oxo-7,8-dihydroguanine and their corresponding oligonucleotides with normal guanine, but piperidine-sensitive damage was induced more frequently in the former than in the latter. Sequence analysis of irradiated oligonucleotides showed that not only 8-oxo-7,8-dihydroguanine but also its neighboring bases and the cytosine residue that is paired to it became piperidine-sensitive in both oligonucleotides. These results suggest that 8-oxo-7,8-dihydroguanine, its neighboring bases and the opposite cytosine are candidates for radiation damage hot spots.     

Limited repair of 8-hydroxy-7,8-dihydroguanine residues in human testicular cells

Oxidative damage in testicular DNA is associated with poor semen quality, reduced fertility and increased risk of stillbirths and birth defects. These DNA lesions are predominantly removed by base excision repair. Cellular extracts from human and rat testicular cells and three enriched populations of rat male germ cells (primary spermatocytes, round spermatids and elongating/elongated spermatids) all showed proficient excision/incision of 5-hydroxycytosine, thymine glycol and 2,6-diamino-4-hydroxy-5-formamidopyrimidine. DNA containing 8-oxo-7,8-dihydroguanine was excised poorly by human testicular cell extracts, although 8-oxoguanine-DNA glycosylase-1 (hOGG1) was present in human testicular cells, at levels that varied markedly between 13 individuals. This excision was as low as with human mononuclear blood cell extracts. The level of endonuclease III homologue-1 (NTH1), which excises oxidised pyrimidines, was higher in testicular than in somatic cells of both species. Cellular repair studies of lesions recognised by formamidopyrimidine-DNA glycosylase (Fpg) or endonuclease III (Nth) were assayed with alkaline elution and the Comet assay. Consistent with the enzymatic activities, human testicular cells showed poor removal of Fpg-sensitive lesions but efficient repair of Nth-sensitive lesions. Rat testicular cells efficiently repaired both Fpg- and Nth-sensitive lesions. In conclusion, human testicular cells have limited capacity to repair important oxidative DNA lesions, which could lead to impaired reproduction and de novo mutations.     

Combination reactions of superoxide with 8-Oxo-7,8-dihydroguanine radicals in DNA: kinetics and end products

One of the major biomarkers of oxidative stress and oxidative damage of cellular DNA is 8-oxo-7,8-dihydroguanine (8-oxoGua), which is more easily oxidized than guanine to diverse oxidative products. In this work, we have investigated further oxidative transformations of 8-oxoGua in single- and double-stranded oligonucleotides to the dehydroguanidinohydantoin, oxaluric acid, and diastereomeric spiroiminodihydantoin lesions. The relative distributions of these end products were explored by a combined kinetic laser spectroscopy and mass spectrometry approach and are shown to depend markedly on the presence of superoxide radical anions. The 8-oxaGua radicals were produced by one-electron oxidation of 8-oxoGua by 2-aminopurine radicals generated by the two-photon ionization of 2-aminopurine residues site specifically positioned in 5'-d(CC[2-aminopurine]TC[8-oxoGua]CTACC). The hydrated electrons also formed in the photoionization process were trapped by dissolved molecular oxygen thus producing superoxide. A combination reaction between the 8-oxoGua and superoxide radicals occurs with the rate constant of (1.3 +/- 0.2) x 10(8) m(-1) s(-1) and (1.0 +/- 0.5) x 10(8) m(-1) s(-1) in single- and double-stranded DNA, respectively. The major end products of this reaction are the dehydroguanidinohydantoin lesions that slowly hydrolyze to oxaluric acid residues. In the presence of Cu,Zn-superoxide dismutase, an enzyme that induces the rapid catalytic dismutation of superoxide to the less reactive H(2)O(2) and O(2), the yields of the dehydroguanidinohydantion lesions become negligible. Under these conditions, the 8-oxoGua radicals do not exhibit any observable reactivities with oxygen (k < 10(2) m(-1) s(-1)), decay on the time interval of several seconds, and the major reaction products are the spiroiminodihydantoin lesions. The possible biological implications of the 8-oxoGua oxidation are discussed.