Discrepancies in the measurement of UVC-induced 8-oxo-2'-deoxyguanosine: implications for the analysis of oxidative DNA damage.
Ultraviolet (UV) light-induced indirect, oxidative damage to DNA has received increasing attention with respect to the mutagenic and carcinogenic effects of solar radiation. An oxidative lesion that has raised particular interest because of its qualitative and quantitative importance is 8-oxo-2'-deoxyguanosine. This deoxynucleoside lesion is most frequently measured by high performance liquid chromatography with electrochemical detection (HPLC-EC) following enzymatic hydrolysis of DNA or as the base equivalent, 8-oxoguanine, by gas chromatography-mass spectrometry (GC-MS) following acid hydrolysis of DNA. We have noted a discrepancy in the literature whereby the levels of 8-oxo-2'-deoxyguanosine measured by HPLC-EC in UVC-irradiated DNA are significantly higher than when 8-oxoguanine is measured by GC-MS. By making use of the availability of both HPLC-EC and stable-isotope dilution GC-MS methodologies in our laboratory we have confirmed the discrepancy noted in the literature by parallel analysis of the same UVC-irradiated calf thymus DNA samples. Furthermore, analysis of the UVC-induced product by UV-visible spectrophotometry, voltammetry and its detection by a monoclonal antibody which recognises 8-oxo-2'-deoxyguanosine strongly suggests that the product is indeed 8-oxo-2'-deoxyguanosine. Partial explanation for this discrepancy could be an inordinate resistance of UVC-irradiated DNA to formic acid hydrolysis. However, we cannot completely exclude the possibility that there is a formic acid-labile species which co-elutes with 8-oxo-2'-deoxyguanosine in enzymatically digested UVC-irradiated DNA. Whether this phenomenon is unique to UV-irradiation damage or occurs with other systems that cause oxidative damage to DNA awaits further investigation. Irrespective of the exact mechanism, there will be significant implications for the analysis of oxidative DNA damage.
Evans
M D
MD
Division of Chemical Pathology, CMHT, Hodgkin Building, University of Leicester, Leicester, LE1 9HN, United Kingdom. mde2@le.ac.uk
Cooke
M S
MS
Podmore
I D
ID
Zheng
Q
Q
Herbert
K E
KE
Lunec
J
J
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000528:radiation effects*; D004249:DNA Damage; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis; D004563:Electrochemistry; D004797:Enzyme-Linked Immunosorbent Assay; D008401:Gas Chromatography-Mass Spectrometry; D018384:Oxidative Stress; D013053:Spectrophotometry; D014466:Ultraviolet Rays
D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D003849:Deoxyguanosine