EVIDENCE FOR SENSITIZATION OF DNA TO OXIDATIVE DAMAGE DURING ISOLATION

The oxidative base lesion 8-oxo-deoxyguanosine (8-oxo-dG) has been ide ntified in DNA isolated from normal tissue and may occur at elevated l evels 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 pronas e E and phenol extraction techniques; native or oxidatively stressed D NA was isolated to determine the validity of each extraction technique for the subsequent determination of 8-oxo-dG. Whilst the yields of DN A were comparable, after pronase E extraction there was no detectable induction of 8-oxo-dG in reextracted naked DNA or peripheral blood mon onuclear cell DNA that had been oxidatively stressed. However, phenol extraction enhanced the basal levels of 8-oxo-dG detected, and also in duced a significant increase in levels of the modified base after expo sure 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 occurr ing during phenol extraction varies with the degree of oxidative damag e already incurred and infers that labile guanine sites generated duri ng oxidative stress may be detected as 8-oxo-dG residues after phenol extraction.