Original articleNucleotide excision repair of oxidised genomic DNA is not a source of urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine
Graphical abstract
Introduction
The non-invasive assessment of oxidative stress, using nucleic acid-, lipid- and protein-derived biomarkers, has a potential use in studying many major diseases, including cancer, cardiovascular disease, neurodegenerative disease and chronic inflammatory disease [1]. In particular, excreted biomarkers of nucleic acid oxidation may have prognostic use in some conditions, such as radiosensitivity [2], mortality in Type 2 diabetes [3], and survival following radiotherapy [4]. The nucleobase oxidation product 8-oxo-7,8-dihydroguanine (8-oxoGua) is of particular interest and importance, largely because of its biological significance, for example as a replicative and transcriptional mutagenic lesion [5], its modulation of gene expression via affecting transcription factor binding, and DNA methylation; and acceleration of telomere shortening (reviewed in [1]). Furthermore, 8-oxoGua is relatively easy to detect using widely available analytical methodology. Different structural forms of this lesion, consisting of the nucleobase itself (8-oxoGua), the ribonucleoside (8-oxoGuo) and the 2’-deoxyribonucleoside (8-oxodGuo), have been detected in the urine of healthy subjects and patients with various pathologies, but predominantly as 8-oxodGuo, rather than 8-oxoGua. The preference to measure 8-oxodGuo derives from an early study which suggested that urinary 8-oxoGua levels are significantly influenced by diet, whereas 8-oxodGuo levels are not [6], hence the latter became the more favoured urinary biomarker of DNA oxidation, coupled with its relative ease of measurement (via electrochemical detection), and its apparent stability against further oxidation, compared to 8-oxoGua [7], [8]. However, more recent evidence refutes diet as a confounding source of urinary 8-oxoGua in mice [9] and humans [10], [11]. It is widely assumed that 8-oxodGuo is derived from the repair of DNA, and is hence a biomarker of DNA oxidation. In fact, the biological origin of urinary 8-oxodGuo is not known, limiting our interpretation and understanding of differences and changes in the excretion of this biomarker.
Several repair processes prevent the persistence of 8-oxoGua in the genome, either by acting on DNA directly, or by sanitisation of the dGTP pools (nuclear and mitochondrial). Those acting on DNA include base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR), nucleotide incision repair (NIR), and proof-reading by DNA polymerases. To sanitise the dGTP pools, Nudix hydrolases catalyse the hydrolysis of 8-oxo-7,8-dihydro-2′-deoxyguanosine triphosphate (8-oxodGTP), which is a potentially mutagenic DNA polymerase substrate, to 8-oxo-7,8-dihydro-2′-deoxyguanosine monophosphate [7], [12]. The latter is not a substrate for re-phosphorylation, and will not be misincorporated. The activity of BER, predominantly via 8-oxoguanine DNA glycosylase (OGG1), is the primary repair process for removing genomic 8-oxoGua and contributes to urinary 8-oxoGua levels. In contrast, the origins of urinary 8-oxodGuo remain unclear although, as described above, significant progress has been made to exclude contribution from diet [9], [11]. We also cited evidence to argue against cell turnover contributing to urinary lesion levels [11]. Critically, the relative importance of DNA repair pathways in generating free 8-oxodGuo is largely unknown, despite over 25 years of its measurement.
Of all the repair pathways acting on DNA, NER [with the corresponding transcription-coupled NER (TC-NER) and global genome NER (GG-NER) pathways] has the potential to generate 8-oxodGuo, from repair-derived, 8-oxoGua-containing oligonucleotides. Subsequent degradation of these lesion-containing oligonucleotides will release free 8-oxodGuo, in a manner analogous to the processing of cyclobutane thymine dimer-containing oligonucleotide repair products [13], [14], [15]. Indeed, there is considerable evidence to support a role for NER (and some evidence for TC-NER [16], [17], [18]), and NER-initiating proteins in the repair of 8-oxodGuo, in various species [19], [20], [21], [22]. We have proposed previously that Nudix hydrolases, via degradation of 8-oxodGTP, or 8-oxodGDP, to 8-oxodGMP, and subsequent de-phosphorylation, are an alternate source of extracellular 8-oxodGuo [12]. Indeed, there is some evidence in the literature to support our hypothesis, and to indicate that dGTP in the nucleotide pools is an important target for oxidants. For example, knock-down of MTH1 (a.k.a. NUDT1) decreases 8-oxodGuo excretion in response to ionising radiation [23] and exogenous expression of MTH1 increases baseline excretion of 8-oxodGuo [24]. Thus the nucleotide pool, and its attendant sanitising activities, would appear to be a credible process for producing urinary 8-oxodGuo [7].
Clearly, determining the exact ‘DNA’ repair origins of this widely measured urinary nucleic acid oxidation product is critical to enable full interpretation of its measurement, and understand which target molecule is being oxidised. In the present study we have assessed directly the whole body contributions of NER (TC-NER and GG-NER) and MTH1 activity on the genomic DNA levels of 8-oxodGuo, and the production of urinary 8-oxodGuo, in selected genetically-modified mice. The data rule out the contribution of NER, which includes TC-NER and GG-NER, to the levels of urinary 8-oxodGuo.
Section snippets
XPA, XPC knock-out and CSB mutant mice
XPA, XPC knock-out and CSB mutant mice (C57Bl/6J background) mice were housed in conventional cages with a 12∶12 h light-dark cycle and had free access to food and water. All animal experiments were approved by the institutional ethics committee on animal care and experimentation at Leiden University Medical Center. Animals were bred at Leiden University Medical Centre using the methodology described in van Oosten et al. [25]. The CSB animals produce a small truncated protein, devoid of
Urinary 8-oxodGuo and 8-oxoGua concentrations in repair-deficient and wild-type mice
The urinary 8-oxodGuo and 8-oxoGua concentrations (9.93±1.2 and 497.0±68.0 nmol/mmol creatinine, respectively) in wild type mice were comparable with those reported elsewhere in the scientific literature and, importantly, creatinine, and specific gravity values did not vary significantly within each genotype over the collection period. As reported elsewhere [33], urinary 8-oxoGua values were significantly greater than urinary 8-oxodGuo values.
Discussion
Measurement of urinary 8-oxodGuo has received extensive study with a literature database comprising of over 925 articles across a period of 28 years, in which it is commonly referred to as biomarker of oxidatively generated damage to DNA, and released via the action of DNA repair. In fact, the exact origins of this lesion continue to remain obscure, although we have shown that contributions from diet and cell turnover are negligible. This is the first report of DNA and urinary 8-oxodGuo and
Acknowledgements
This project was supported, in part, by ECNIS (Environmental Cancer Risk, Nutrition and Individual Susceptibility), a network of excellence operating within the European Union 6th Framework Program, Priority 5: “Food Quality and Safety” (Contract no. 513943). The following authors were members of ECNIS: MDE, MSC, VM, RS, DG, RR, AS, DHP, PBF, RO.
The authors thank Dr. L. Barregård, Department of Occupational & Environmental Medicine, University of Gothenburg, Sweden, for advice regarding
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2019, Free Radical Biology and MedicineCitation Excerpt :Therefore, it is mainly a measure of generalized intracellular oxidative stress. Considering only 8-oxodG or 8-oxoGuo levels in plasma and urine may result in incomplete estimation of oxidatively modified guanine components since 8-oxo-7,8-dihydroguanine (8-oxoGua), the expected released product from DNA through the base excision repair (BER) pathway has been shown to be present in much higher amounts than 8-oxodG in urine [38–41]. There are several reports using a single plasma 8-oxodG value as a marker of “oxidative stress to nucleic acids”.
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- 1
Present address: School of Allied Health Sciences, Faculty of Health and Life Sciences, De Montfort University, The Gateway, Leicester LE1 9BH, United Kingdom.
- 2
Present address: Analytical & Environmental Sciences Division, Franklin Wilkins-Building, King's College London, London SE1 9NH, United Kingdom.
- 3
Present address: Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford University, Oxford OX3 9DS, United Kingdom.
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Present address: Oxidative Stress Group, Department of Environmental & Occupational Health, Florida International University, Miami, FL 33199, USA.