Evidence for sensitisation of DNA to oxidative damage during isolation.
The oxidative base lesion 8-oxo-deoxyguanosine (8-oxo-dG) has been identified in DNA isolated from normal tissue and may occur at elevated levels during disease. However, the use of phenol during DNA extraction may artificially elevate the detected levels of this lesion. Herein, we have performed a comparative methodological study using both pronase E and phenol extraction techniques; native or oxidatively stressed DNA was isolated to determine the validity of each extraction technique for the subsequent determination of 8-oxo-dG. Whilst the yields of DNA were comparable, after pronase E extraction there was no detectable induction of 8-oxo-dG in reextracted naked DNA or peripheral blood mononuclear cell DNA that had been oxidatively stressed. However, phenol extraction enhanced the basal levels of 8-oxo-dG detected, and also induced a significant increase in levels of the modified base after exposure to oxidative stress. The latter was dependent on the presence of foetal calf serum in the extracellular medium. We have confirmed that phenol extraction sensitises native DNA to subsequent oxidative damage. In addition, this work shows that the extent of sensitisation occurring during phenol extraction varies with the degree of oxidative damage already incurred and infers that labile guanine sites generated during oxidative stress may be detected as 8-oxo-dG residues after phenol extraction.
Division of Chemical Pathology, University of Leicester, Centre for Mechanisms of Human Toxicity, UK.
M T
MT
Finnegan
K E
KE
Herbert
M D
MD
Evans
H R
HR
Griffiths
J
J
Lunec
D000080242:8-Hydroxy-2'-Deoxyguanosine; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000302:isolation & purification* / Q000378:metabolism; D003849:Deoxyguanosine / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D005720:Gamma Rays; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D018384:Oxidative Stress; D019800:Phenol; D010636:Phenols / Q000494:pharmacology; D011402:Pronase / Q000378:metabolism; D013950:Thymus Gland / Q000737:chemistry
D010636:Phenols; D019800:Phenol; D000080242:8-Hydroxy-2'-Deoxyguanosine; D004247:DNA; D006861:Hydrogen Peroxide; D011402:Pronase; D003849:Deoxyguanosine