Phenol isolation of DNA yields higher levels of 8-oxodeoxyguanosine compared to pronase E isolation

Abstract

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) ...

DNA oxidation matters: The HPLC–electrochemical detection assay of 8-oxo-deoxyguanosine and 8-oxo-guanine

“…The addition of a phenol extraction step to this method increased the concentration of oxo 8 dG by an amount that was modest in absolute terms (0.25 oxo 8 dG͞10 5 dG) but statistically significant. In contrast, the value we obtained with an alternative phenol-free method (22,28) was five times higher than that obtained with the chaotropic NaI method. Moreover, this latter phenol-free value was unaffected by the addition of a phenol extraction step.…”

“…The validity of the standard HPLC-EC-based oxo 8 dG method (25,27,30) often has been questioned, and modifications of the technique have been introduced (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). Much of the debate has surrounded the use of phenol (15,18,19,22,31) after a report (14) that phenol is a prooxidant during DNA extraction. Although we have never found phenol to cause a large increase in oxo 8 dG, we have reinvestigated its use by adding a phenol-extraction step to phenol-free methods.…”

“…Consequently, to test the hypothesis that NaI in the chaotropic method had artifactually depressed the measured level of oxo 8 dG (e.g., by chemical decomposition), we substituted sodium acetate for NaI in the chaotropic ϩ phenol method and found that the value remained unchanged (0.50 Ϯ 0.06 oxo 8 dG͞10 5 dG). Another method (22,28), the hydrolysis of samples with pronase, increased the ratio to 1.2 Ϯ 0.03 oxo 8 dG͞10 5 dG (P Ͻ 0.03) and resulted in greater variability. By using rat liver nuclei from young animals, we found that the modified chaotropic NaI procedure (see Methods) produced the lowest baseline values (0.04 Ϯ 0.002 oxo 8 dG͞10 5 dG).…”

“…The fourth protocol was (iv) the phenol extraction procedure previously used in our laboratory (25). Two final procedures were based on prolonged (overnight) pronase incubation, as described (22,28): (v) an overnight pronase digestion, and (vi) an overnight pronase digestion with the addition of a phenol extraction step. In all six procedures the DNA was dried by centrifugation under vacuum before digestion and the samples were analyzed immediately.…”

“…At the same time, however, modifications and alternatives to the initial HPLC-EC techniques have been introduced, leading to discordant results, the identification of various pitfalls, and disagreements about the most appropriate technique (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23). Because of the broad interest in using oxo 8 dG as a biomarker, it is critical to identify a common set of methods that minimize error in measurement of the adduct.…”