Nucleotide excision repair of oxidised genomic DNA is not a source of urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine

Abstract

Urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) is a widely measured biomarker of oxidative stress. It has been commonly assumed to be a product of DNA repair, and therefore reflective of DNA oxidation. However, the source of urinary 8-oxodGuo is not understood, although potential confounding contributions from cell turnover and diet have been ruled out. Clearly it is critical to understand the precise biological origins of this important biomarker, so that the target molecule that is oxidised can be identified, and the significance of its excretion can be interpreted fully. In the present study we aimed to assess the contributions of nucleotide excision repair (NER), by both the global genome NER (GG-NER) and transcription-coupled NER (TC-NER) pathways, and sanitisation of the dGTP pool (e.g. via the activity of the MTH1 protein), on the production of 8-oxodGuo, using selected genetically-modified mice. In xeroderma pigmentosum A (XPA) mice, in which GG-NER and TC-NER are both defective, the urinary 8-oxodGuo data were unequivocal in ruling out a contribution from NER. In line with the XPA data, the production of urinary 8-oxodGuo was not affected in the xeroderma pigmentosum C mice, specifically excluding a role of the GG-NER pathway. The bulk of the literature supports the mechanism that the NER proteins are responsible for removing damage to the transcribed strand of DNA via TC-NER, and on this basis we also examined Cockayne Syndrome mice, which have a functional loss of TC-NER. These mice showed no difference in urinary 8-oxodGuo excretion, compared to wild type, demonstrating that TC-NER does not contribute to urinary 8-oxodGuo levels. These findings call into question whether genomic DNA is the primary source of urinary 8-oxodGuo, which would largely exclude it as a biomarker of DNA oxidation. The urinary 8-oxodGuo levels from the MTH1 mice (both knock-out and hMTH1-Tg) were not significantly different to the wild-type mice. We suggest that these findings are due to redundancy in the process, and that other enzymes substitute for the lack of MTH1, however the present study cannot determine whether or not the 2'-deoxyribonucleotide pool is the source of urinary 8-oxodGuo. On the basis of the above, urinary 8-oxodGuo is most accurately defined as a non-invasive biomarker of oxidative stress, derived from oxidatively generated damage to 2'-deoxyguanosine.

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“…Its precise origin is not fully documented, and diet and cell turnover have been ruled out (Evans et al 2016). The authors could not establish whether 8-oxodG derives from the 2′-deoxyribonucleotide pool, and conclude that: “8-ox-odGuo is most accurately described as a non-invasive biomarker of oxidative stress derived from oxidatively generated damage to 2′-deoxyguanosine.” It should be further noted that for obvious reasons (ease of use and low cost), 8-oxodG in urine is often measured with commercially available ELISA kits and this includes almost all studies mentioned above.…”

MTH1 as a Chemotherapeutic Target: The Elephant in the Room

“…In this regard, it was recently reported that the loss of the Von-Hippel Lindau (VHL) factor, a tumor suppressor and E3 ubiquitin ligase that degrades the hypoxia-inducible factor HIF under normoxic conditions, sensitizes cells to MTH1 inhibition [49]. The unaltered mutagenic burden in MTH1-null mice [28] and the lack of difference in genomic 8-oxodG processing byproducts, such as 8-oxo-7,8-dihydro-2′-deoxyguanosine, in MTH1-null vs. wild-type animals [50] potentially point to redundant mechanisms that can compensate for MTH1 loss. Alternatively, enhanced antioxidant pathways, such as Nrf2 [51], could mitigate the need for MTH1 function by reducing ROS levels and, thus, oxidation in the nucleotide pool.…”

Formation and repair of oxidatively generated damage in cellular DNA

“…The measurement of 8-oxoG and 8-oxodGuo, which is free of methodological artefactual oxidation, in human and animal fluids by HPLC-MS/MS will not be discussed in detail in this review (for recent reviews, see [111,171,172]. The origin of released 8-oxodG in urine still remains unclear particularly with purines since their metabolism involves enzymatic oxidation [182]. As a final remark, one should note the lack of accurate data on the formation of oxidatively induced damage to 2-deoxyribose in cellular DNA.…”