Urinary 8-oxo-2′-deoxyguanosine:Redox regulation of DNA repair in vivo?

DNA is susceptible to damage by reactive oxygen species (ROS). ROS are produced during normal and pathophysiological processes in addition to ionizing radiation, environmental mutagens, and carcinogens. 8-oxo-2′-deoxyguanosine (8-oxodG) is probably one of the most abundant DNA lesion formed during oxidative stress. This potentially mutagenic lesion causes G → T transversions and is therefore an important candidate lesion for repair, particularly in mammalian cells. Several pathways exist for the removal, or repair, of this lesion from mammalian DNA. The most established is via the base excision repair enzyme, human 8-oxoguanine glycosylase (hOgg1), which acts in combination with the human apurinic endonuclease (hApe). The latter is known to respond to regulation by redox reactions and may act in combination with hOgg1. We discuss evidence in this review article concerning alternative pathways in humans, such as nucleotide excision repair (NER), which could possibly remove the 8-oxodG lesion. We also propose that redox-active components of the diet, such as vitamin C, may promote such repair, affecting NER specifically. © 2002 Elsevier Science Inc.

Development of a quality control material for the measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine, an in vivo marker of oxidative stress, and comparison of results from different laboratories

The measurement of 8-oxo-7,8-dihydro-2'-deoxyguanosine is an increasingly popular marker of in vivo oxidative damage to DNA. A random-sequence 21-mer oligonucleotide 5'-TCA GXC GTA CGT GAT CTC AGT-3' in which X was 8-oxo-guanine (8-oxo-G) was purified and accurate determination of the oxidised base was confirmed by a 32P-end labelling strategy. The lyophilised material was analysed for its absolute content of 8-oxo-dG by several major laboratories in Europe and one in Japan. Most laboratories using HPLC-ECD underestimated, while GC-MS-SIM overestimated the level of the lesion. HPLC-ECD measured the target value with greatest accuracy. The results also suggest that none of the procedures can accurately quantitate levels of 1 in 10(6) 8-oxo-(d)G in DNA.

Novel repair action of vitamin C upon in vivo oxidative DNA damage

There appears to be a paucity of data examining the effect of dietary antioxidants on levels of oxidative DNA damage in vivo, limiting evidence-based assessment of antioxidant efficacy, mechanisms and recommendation for optimal intake. We have examined levels of 8-oxo-2'-deoxyguanosine (8-oxodG) in mononuclear cell DNA, serum and urine from subjects undergoing supplementation with 500 mg/day vitamin C. Significant decreases in DNA levels of 8-oxodG were seen, correlating strongly with increases in plasma vitamin C concentration. Furthermore we established a timecourse for sequential, significant increases in serum and urinary 8-oxodG levels. These results illustrate, for the first time in humans, the kinetics of 8-oxodG removal and processing in vivo, suggesting a role for vitamin C in the regulation of DNA repair enzymes and thereby demonstrating a non-scavenging antioxidant effect.

Evidence for sensitisation of DNA to oxidative damage during isolation

The oxidative base lesion 8-oxo-deoxyguanosine (8-oxo-dG) has been identified in DNA isolated from normal tissue and may occur at elevated levels during disease. However, the use of phenol during DNA extraction may artificially elevate the detected levels of this lesion. Herein, we have performed a comparative methodological study using both pronase E and phenol extraction techniques; native or oxidatively stressed DNA was isolated to determine the validity of each extraction technique for the subsequent determination of 8-oxo-dG. Whilst the yields of DNA were comparable, after pronase E extraction there was no detectable induction of 8-oxo-dG in reextracted naked DNA or peripheral blood mononuclear cell DNA that had been oxidatively stressed. However, phenol extraction enhanced the basal levels of 8-oxo-dG detected, and also induced a significant increase in levels of the modified base after exposure to oxidative stress. The latter was dependent on the presence of foetal calf serum in the extracellular medium. We have confirmed that phenol extraction sensitises native DNA to subsequent oxidative damage. In addition, this work shows that the extent of sensitisation occurring during phenol extraction varies with the degree of oxidative damage already incurred and infers that labile guanine sites generated during oxidative stress may be detected as 8-oxo-dG residues after phenol extraction.

Novel monoclonal antibody recognition of oxidative DNA damage adduct, deoxycytidine-glyoxal

Glyoxal, a reactive aldehyde, is a decomposition product of lipid hydroperoxides, oxidative deoxyribose breakdown, or autoxidation of sugars, such as glucose. It readily forms DNA adducts, generating potential carcinogens such as glyoxalated deoxycytidine (gdC). A major drawback in assessing gdC formation in cellular DNA has been methodologic sensitivity. We have developed an mAb that specifically recognizes gdC. Balb/c mice were immunized with DNA, oxidatively modified by UVC/hydrogen peroxide in the presence of endogenous metal ions. Although UVC is not normally considered an oxidizing agent, a UVC/hydrogen peroxide combination may lead to glyoxalated bases arising from hydroxyl radical damage to deoxyribose. This damaging system was used to induce numerous oxidative lesions including glyoxal DNA modifications, from which resulted a number of clones. Clone F3/9/H2/G5 showed increased reactivity toward glyoxal-modified DNA greater than that of the immunizing antigen. ELISA unequivocally showed Ab recognition toward gdC, which was confirmed by gas chromatography-mass spectrometry of the derivatized adduct after formic acid hydrolysis to the modified base. Binding of Ab F3/9 with glyoxalated and untreated oligomers containing deoxycytidine, deoxyguanosine, thymidine, and deoxyadenosine assessed by ELISA produced significant recognition (p 0.0001) of glyoxal-modified deoxycytidine greater than that of untreated oligomer. Additionally, inhibition ELISA studies using the glyoxalated and native deoxycytidine oligomer showed increased recognition for gdC with more than a 5-fold difference in IC50 values. DNA modified with increasing levels of iron (II)/EDTA produced a dose-dependent increase in Ab F3/9 binding. This was reduced in the presence of catalase or aminoguanidine. We have validated the potential of gdC as a marker of oxidative DNA damage and showed negligible cross-reactivity with 8-oxo-2'-deoxyguanosine or malondialdehyde-modified DNA as well as its utility in immunocytochemistry. Formation of the gdC adduct may involve intermediate structures; however, our results strongly suggest Ab F3/9 has major specificity for the predominant product, 5-hydroxyacetyl-dC.

Chemical synthesis of DNA and RNA containing thio-substituted bases and their post-synthetic modifications

Modified oligonucleotides containing sulphur group have been useful tools for studies of carcinogenesis, protein or nucleic acid structures and functions, protein-nucleic acid interactions, and for antisense modulation of gene expression. One successful example has been the synthesis and study of oligodeoxynucleotides containing 6-thio-2'-deoxyguanine. 6-Thio-2-deoxyguanosine was first discovered as metabolic compound of 6- mercaptopurine (6-MP). Later, it was applied as drug to cure leukaemia. During the research of its toxicity, a method was developed to use the sulphur group as a versatile position for post-synthetic modification. The advantage of application of post-synthetic modification lies in its convenience. Synthesis of oligomers with normal sequences has become routine work in most laboratories. However, design and synthesis of a proper phosphoramidite monomer for a new modified nucleoside are always difficult tasks even for a skilful chemist. Thus an alternative method (post-synthetic method) has been invented to overcome the difficulties. This was achieved by incorporation of versatile nucleotides into oligomers which contain a leaving group, that is sufficiently stable to withstand the conditions of synthesis but can be substituted by nucleophiles after synthesis, to produce, a series of oligomers each containing a different modified base. In the current project, a phosphoramidite monomer with 6-thioguanine has been successfully synthesised and incorporated into RNA. A deprotection procedure, which is specific for RNA was designed for oligomers containing 6-thioguanosine. The results were validated by various methods (UV, HPLC, enzymatic digestion). Pioneer work in utilization of the versatile sulphur group for post-synthetic modification was also tested. Post-synthetic modification was also carried out on DNA with 6- deoxythioguanosine. Electrophilic reagents with various functional groups (alphatic, aromatic, fluorescent) and bi-functional groups have been attached with the oligomers.

Chemical synthesis of isotopically labelled (M+4) purine nucleosides and their incorporation into DNA oligomers

Development of accurate and sensitive analytical methods to measure the level of biomarkers, such as 8-oxo-guanine or its corresponding nucleoside, 8-oxo-2’-deoxyguanosine, has become imperative in the study of DNA oxidative damage in vivo. Of the most promising techniques, HPLC-MS/MS, has many attractive advantages. Like any method that employs the MS technique, its accuracy depends on the use of multiply, isotopically-labelled internal standards. This project is aimed at making available such internal standards. The first task was to synthesise the multiply, isotopically-labelled bases (M+4) guanine and (M+4) 8-oxo-guanine. Synthetic routes for both (M+4) guanine and (M+4) 8-oxo-guanine were designed and validated using the unlabelled compounds. The reaction conditions were also optimized during the “dry runs”. The amination of the 4-hydroxy-2,6-dichloropyrimidine, appeared to be very sensitive to the purity of the commercial [15]N benzylamine reagent. Having failed, after several attempts, to obtain the pure reagent from commercial suppliers, [15]N benzylamine was successfully synthesised in our laboratory and used in the first synthesis of (M+4) guanine. Although (M+4) bases can be, and indeed have been used as internal standards in the quantitative analysis of oxidative damage, they can not account for the errors that may occur during the early sample preparation stages. Therefore, internal standards in the form of nucleosides and DNA oligomers are more desirable. After evaluating a number of methods, an enzymatic transglycolization technique was adopted for the transfer of the labelled bases to give their corresponding nucleosides. Both (M+4) 2-deoxyguanosine and (M+4) 8-oxo-2’-deoxyguanosine can be purified on micro scale by HPLC. The challenge came from the purification of larger scale (>50 mg) synthesis of nucleosides. A gel filtration method was successfully developed, which resulted in excellent separation of (M+4) 2’-deoxyguanosine from the incubation mixture. The (M+4) 2’-deoxyguanosine was then fully protected in three steps and successfully incorporated, by solid supported synthesis, into a DNA oligomer containing 18 residues. Thus, synthesis of 8-oxo-deoxyguanosine on a bigger scale for its future incorporation into DNA oligomers is now a possibility resulting from this thesis work. We believe that these internal standards can be used to develop procedures that can make the measurement of oxidative DNA damage more accurate and sensitive.