Background and Radiation-induced 8-hydroxy-2′-deoxyguanosine in γ-irradiatedEscherichia Coli

Abstract

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.

Singlet Oxygen Induces Oxidation of Cellular DNA

“…3). A nonsignificant variation in the level of unlabeled 8-oxodGuo and an important formation of 18 O-labeled 8-oxodGuo is measured when cells were incubated with the labeled endoperoxide. The results, summarized in Fig.…”

“…Thereafter, the cells were collected by centrifugation to remove the excess of DHPN 18 O 2 and resuspended in PBS buffer before incubation at 37°C. Under these conditions (Fig.…”

“…In contrast, 8-oxodGuo was found to be the major oxidation product formed upon exposure of isolated DNA to 1 O 2 (6,11,16). However, 8-oxodGuo cannot be considered as a specific biological marker of 1 O 2 , since this DNA lesion could be formed under various conditions of oxidative stress, including those generated by one-electron process (17), hydroxyl radical (18), and Fenton-type reactions (19). On one hand, this explains why 8-oxodGuo could be used as an ubiquitous biomarker of DNA oxidation (20 -22).…”

“…18 O 2 were added. For control experiment, complete deactivation of DHPNO 2 was obtained by heating the endoperoxide for 40 min at 70°C.…”

“…Emphasis was placed on the determination of the mechanism of DNA oxidation. For this purpose, the 18 O-labeled endoperoxide DHPN 18 O 2 was prepared to assess if the formation of oxidative damage results from the direct reaction of 1 O 2 within cellular DNA. It was clearly shown that intracellular formation of singlet oxygen is able to efficiently produce 8-oxodGuo.…”