Ozonation of DNA Forms Adducts: A ³²P-DNA Labeling and Thin-layer Chromatography Technique to Measure DNA Environmental Biomarkers

Abstract

Little direct documented evidence of ozone's genotoxicity exists. Deoxyribonucleic acid (DNA) adducts are produced by environmental toxic agents, including ozone. We have described a modified thin-layer chromatography (TLC) technique that can assess adduct formation as a biomarker of ozone injury. This requires ³²P-labeling DNA, digestion of deoxynucleotides (dNMPs), and separation in two-dimensional PEI-cellulose TLC. We have applied this technique to control DNAs, to control DNA in solution exposed to acute ambient ozone, and to control DNA exposed to acute bubbled-through ozone (2 ppm for 24 h). We detected stable DNA adducts, including hydroxymethyluracil (HMU), thymine glycol (TC), 8-hydroxyguanine (8–OHG), and demonstrated, as yet, unidentified adducts that may serve as a “fingerprint” pattern of DNA adduction. This technique quantifies low-molecularmass DNA adducts, both in vivo and in vitro, with potential applications to environmental toxicology.