Background and radiation-induced 8-hydroxy-2'-deoxyguanosine in gamma-irradiated Escherichia coli

The DNA base damage product, 8-hydroxy-2'-deoxyguanosine (7-hydro-8-oxo-2'-deoxyguanosine, or 8-OHdG) was measured in 2'-deoxyribonucleoside digests of DNA from irradiated E. coli or model DNA using HPLC separation and electrochemical detection (LCED). The LCED technique enabled the specific quantitation of 25 fmol of 8-OHdG (signal: noise ratio = 4:1) among a variety of DNA base lesions within the sample. A radiation dose-yield relationship for 8-OHdG was observable in cells that were rapidly lysed after irradiation using a chloroform-saturated, sarkosyl and EDTA solution at 4 degrees C (Tilby and Loverock 1983). The observed yield of 8-OHdG was 7.05 +/- 0.27 pmol J-1 and was against a background of 120 8-OHdG per E. coli genome. This yield translates to an average of 12 additional 8-OHdG lesions per genome at the mean lethal dose. The low radiochemical yield and high background suggests that factors other than the toxicity of 8-OHdG per se--such as its participation in clusters of base lesions (e.g. Ward 1985)--must contribute significantly to cell killing. Although exposure of DNA to phenol mixtures has often been reported to increase 8-OHdG, we found that a more significant effect of phenol mixtures was the potentiation of the radiochemical yields of 8-OHdG in model DNA from 5.26 +/- 0.48 to 158 +/- 12.2 nmol J-1. Other factors, such as the exposure of dried 2'-deoxyribonucleoside digests to ambient humidity in the presence of buffer components, were shown to contribute more significantly to the background of 8-OHdG.