Phenol sensitization of DNA to subsequent oxidative damage in 8-hydroxyguanine assays.
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.
Claycamp
H G
HG
Department of Environmental and Occupational Health, University of Pittsburgh, PA 15261.
D015415:Biomarkers; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry / Q000187:drug effects* / Q000302:isolation & purification; D004249:DNA Damage; D006147:Guanine / Q000031:analogs & derivatives* / Q000032:analysis; D008722:Methods; D019800:Phenol; D010636:Phenols / Q000494:pharmacology*
D015415:Biomarkers; D010636:Phenols; D019800:Phenol; C024829:8-hydroxyguanine; D006147:Guanine; D004247:DNA