DNA Adduct Quantification in Carcinogen-Treated Cells

DNA was isolated from the cellular pellets by a chloroform/phenol extraction method (50 (link)). The amount of DNA recovered from each carcinogen treatment was determined by UV spectroscopy, assuming a concentration of DNA (50 μg/mL) is equal to 1.0 absorbance unit at 260 nm. Isotopically labeled internal standards of each DNA adduct were added to the DNA recovered from the treated hepatocytes at a level of 1 adduct per 10⁶ DNA bases. The DNA from the respective time points of the individually treated 4-ABP and HAA hepatocyte samples were then pooled. The enzymatic digestion conditions used for the hydrolysis of DNA (~2 – 10 μg) in 5 mM Bis-Tris-HCl buffer (pH 7.1, 50 μL) employed DNAse I for 1.5 h, followed by incubation with nuclease P1 for 3 h, and then digested with alkaline phosphatase and phosphodiesterase for 18 h (45 (link)). These enzyme digestion conditions were shown to be highly efficient in the recovery of the dG-C8 adducts of PhIP, MeIQx, IQ, and 4-ABP from calf thymus DNA modified with these carcinogens (42 (link)–45 (link),51 (link),52 (link)). The DNA adducts were purified by solid phase extraction, using HyperSep^™ filter SpinTips, as previously described (52 (link),53 (link)).