Phenol sensitization of DNA to subsequent oxidative damage in 8-hydroxyguanine assays

Abstract

The DNA base adduct, 8-hydroxyguanine (8-OHGua), has been reported to be a key biomarker relevant to carcinogenesis and cellular oxidative stress important in tumor promotion. Although investigators often report artificially high levels of 8-OHGua in DNA samples that have been exposed to phenol solutions and/or air during processing, few quantitative results are available. We show that routine phenol-based DNA purification procedures can increase 8-hydroxydeoxyguanosine (8-OHdG) levels 20-fold in samples that are exposed to air after the phenol is removed from the solutions. Surprisingly, air exposure alone accounts for a significant portion of this increase (4-fold) when compared to dG or DNA samples that have been solubilized in buffers purged with nitrogen. Most importantly, phenol treatments of DNA are shown to sensitize DNA to 8-OHdG formation by subsequent exposures to air. The sensitization of DNA occurs even though extensive dialysis is used between phenol treatment and enzymatic DNA digestion. Alternate procedures, including chloroform:isoamyl-alcohol extractions, also yield air-sensitive DNA samples. Other artifacts of organic extraction prior to air exposure include alterations in DNA base ratios after nuclease digestions. Overall, these results strongly suggest that studies of 8-OHdG in carcinogenesis should avoid dry conditions, such as lyophilization followed by exposure to air, and that all four of the bases should be monitored before 8-OHdG concentrations are normalized by undamaged deoxynucleoside concentrations. Failure to heed these precautions can lead to 2- to 20-fold overestimates of 8-OHdG in target tissues or in vitro models.

Size and topology of exogenous DNA as determinant factors of transgene transcription in mammalian cells

“…[12][13][14][15] The former induces strand breaks and the latter oxidizes DNA. It was reported that DNA recovered from a gel visualized by EtBr plus light was a poor substrate for in vitro transcription.…”

“…It is known that light irradiation in the presence of ethidium bromide (EtBr), organic solvent extractions, and UV-irradiation damage DNA, [12][13][14][15][16] and that the formation of DNA lesions results in transcriptional inhibition and errors. [17][18][19][20] Thus, we purified closed circular molecules and linearized DNAs in the same gel to compare the effect of the topology precisely.…”

Suppression of Lipid Hydroperoxide-Induced Oxidative Damage to Cellular DNA by Esculetin

“…[23][24][25] Phenol and atmospheric oxygen are proposed to be responsible for the artificial formation of 8-oxodG during these procedures. In this study, incubation with enzymes during the isolation and hydrolysis of DNA was performed under an argon atmosphere.…”

Aging and the Accumulation of Oxidative Damage to DNA

“…Because LC/MS/MS and LC/MS do not require sample derivatization for assay, the oxidation of DNA is suppressed [34]. On the other hand, in the case of HPLC/ECD, a significant amount of oxidative damage can be induced during the isolation of DNA, especially in the presence of phenol [40] . Phenol, a reducing agent, is thought to reduce metal ions (e. g., iron ions) present in biological extracts .…”