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Oxidative damage is thought to play a role in the aetiology of aging and a number of diseases including cancer, chronic inflammation, ischemia, degenerative arterial and autoimmune diseases [I]. 8-oxodeoxyguanosine (8-oxodG), an oxidative DNA adduct. has gained much popularity as a biomarker of damage to DNA. HPLC combined with electrochemical detection provides a selective and sensitive method of measuring 8-oxodG [2]. It has been reported that phenol extraction of DNA may cause sensitization of DNA to subsquent oxidative damage and result in higher 8-oxodG levels [3]. In this study we compare phenol isolation with pronase. E isolation of DNA [4].Human peripheral blood mononuclear cells (PBMC) were isolated from whole blood using Histopaque 1077 and treated using two different exposure protocols to induce oxidative stress. First, the mononuclear cells were treated on ice, to reduce DNA repair, with H202 (400vM) for 15min. Second, PBMC were exposed on ice to a Vickrad ' %o source, and treated with gamma irradiation at a rate of 0.48Gylsec (OGy, 2OGy, 20OGy). Mononuclear cell DNA was extracted using the pronase E method of Kendall et al, [4] or the phenol method based on a procedure described by Winyard et al [S]. DNA was enzymatically digested to the deoxynucleoside level [6]; micrococcal nuclease (0.14units/4pg DNA) was used in place of N.crussa endonuclease. The method for reversedphase HPLC of the DNA deoxynucleosides was modified from the procedure of Floyd et a1 [7].The levels of DNA extracted using either method were not significantly different (data not shown). The results in Figure 1 show that lower levels of 8-oxodG were observed in H202 treated and untreated PBMC and naked calf thymus DNA when DNA was isolated using the pronase E method compared to the phenol method. In the experiments where the cells were treated with hydrogen peroxide in the presence of foetal calf serum (FCS) a response to treatment with H202 was observed (p< 0.05) in that 8-oxodG levels were increased but only for phenol extraction of DNA (Figure 1). Similarly for y-irradiated cells the levels of 8-oxodG observed using pronase E extraction were lower than the levels observed using the phenol method (p< 0.05) (Figure 2). A dose response was suggested using phenol isolation from yirradiated PBMC 0-2OOGy (Figure 2). Higher levels of 8-oxodG were observed on extraction of naked calf thymus DNA using the phenol method (p< 0.05) but no increase in 8-oxodG proportional to increased irradiation was seen for phenol extracts of DNA.The importance of establishing a reliable method to measure Abbreviations used: 8-oxodG, 8-oxodeoxyguanosine; HPLC, high performance liquid chromatography; PBMC, peripheral blood mononuclear cells; FCS, foetal calf serum; H202, hydrogen peroxide; Gy, Gray; N.crassa, neurospora crassa. Pronase E Phenol Figurc 1: Tlus graph show the lewls of 8-0xodG in PBMC or naked DNA treated wthJOOph4 hvlrogcn pcroude tor ISmin +SEM for three scp.ratc experiments on ice tsEh d u e represents the mean 20, (DNA) (PHMC) (DNA) (PHMC) ...

Development of an assay to measure thxoguanine using HPLC with electrochemical detection.