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

• Published in: Carcinogenesis (New York) Vol. 13; no. 7; p. 1289
• Main Author: Claycamp, H G
• Format: Journal Article
• Language: English
• Published: England 01-07-1992
• Subjects: Biomarkers; Chromatography, High Pressure Liquid; DNA - chemistry; DNA - drug effects; DNA - isolation & purification; DNA Damage; Guanine - analogs & derivatives; Guanine - analysis; Methods; Phenol; Phenols - pharmacology
• ISSN: 0143-3334
• DOI: 10.1093/carcin/13.7.1289

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.