Journal Article

SVPD-post-labeling detection of oxidative damage negates the problem of adventitious oxidative effects during <sup>32</sup>P-labeling

George D.D. Jones, Lynda Dickinson, Joseph Lunec and Michael N. Routledge

in Carcinogenesis

Volume 20, issue 3, pages 503-507
Published in print March 1999 | ISSN: 0143-3334
Published online March 1999 | e-ISSN: 1460-2180 | DOI:
SVPD-post-labeling detection of oxidative damage negates the problem of adventitious oxidative effects during 32P-labeling

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The exploitation of oxidative DNA lesions as biomarkers of oxidative stress in vivo requires techniques that allow for the precise and valid measurement of oxidative damage to DNA. Previously, endogenous levels of the oxidative lesion 8-hydroxy-2′-deoxyguanosine (8-HO-dG) in rat tissues determined by a micrococcal nuclease/calf spleen phosphodiesterase-based 32P-post-labeling protocol were found to be at least 10-fold higher than those determined by HPLC with electrochemical detection. This was attributed to the adventitious oxidation of the normal nucleotides (dGp) occurring during the labeling stage of the post-labeling protocol, which could only be prevented by the introduction of additional chromatographic steps to remove the unmodified species prior to labeling. In the present study we report that an alternative snake venom phosphodiesterase-based 32P-post-labeling procedure (SVPD-post-labeling) negates the problem of adventitious oxidative damage during labeling by virtue of a unique digestion strategy. In SVPD-post-labeling, digestion yields certain lesions (thymine glycols, phosphoglycolates and abasic sites) as damage-containing dimer species which are ready substrates for labeling. In contrast, the undamaged DNA is recovered as mononucleoside species (dN) which are not substrates for labeling and so remain undetected. Furthermore, even if the mononucleosides are oxidized during labeling, they will not contribute to the level of damage detected. Indeed, we demonstrate that neither the external γ-irradiation of the digested DNA samples nor increasing the incubation time of the labeling reaction alters the levels of damage detected by SVPD-post-labeling. The negation of adventitious oxidative effects during labeling deems that an optimized SVPD-post-labeling procedure should be well-suited for the biomonitoring of endogenous oxidative stress in vivo.

Keywords: 8-HO-dGp, 8-hydroxy-2′-deoxyguanosine-3′-monophosphates; 8-OH-G, 8-hydroxyguanine; 8-OH-dG, 8-hydroxy-2′-deoxyguanosine; CSPD, calf spleen phosphodiesterase; dG, 2′-deoxyguanosine; dGp, 2′-deoxyguanosine-3′-monophosphates; dN, 2′-deoxynucleosides; dNp, 2′-deoxynucleoside-3′-monophosphates; GC–MS/SIM, gas chromatography–mass spectroscopy with selective ion monitoring; HO-, hydroxyl moieties; HPLC–ECD, high performance liquid chromatography with electrochemical detection; MN, micrococcal nuclease; OH˙, hydroxyl radicals; 32P-HPLC, high performance liquid chromatography with radioactivity detection; PAGE, polyacrylamide gel electrophoresis; pdN, 2′-deoxynucleoside 5′-monophosphates; ROS, reactive oxygen species; SAP, shrimp alkaline phosphatase; SVPD, snake venom phosphodiesterase; T4PNK, T4 polynucleotide kinase; TLC, thin-layer chromatography.

Journal Article.  3663 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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