SVPD-post-labeling detection of oxidative damage negates the problem of adventitious oxidative effects during 32P-labeling.
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 postlabeling 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-postlabeling) 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 gamma-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.
Jones
G D
GD
Biomolecular Damage Group, Centre for Mechanisms of Human Toxicity, University of Leicester, UK. gdj2@leicester.ac.uk
Dickinson
L
L
Lunec
J
J
Routledge
M N
MN
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004249:DNA Damage; D004563:Electrochemistry; D018384:Oxidative Stress; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry; D010759:Phosphorus Isotopes; D051381:Rats; D012910:Snake Venoms / Q000201:enzymology
D010759:Phosphorus Isotopes; D012910:Snake Venoms; D010727:Phosphoric Diester Hydrolases