Quantitative high-performance liquid chromatography analysis of DNA oxidized in vitro and in vivo

Abstract

Oxidative modification of genetic material has been implicated as a factor in carcinogenesis, particularly during promotion and progression, and therefore there is a need for sensitive detection of oxidized DNA bases. We developed a method that can be applied to DNA isolated from any source and used to simultaneously quantify oxidized nucleosides without a need to prelabel the DNA or use destructive hydrolytic procedures. This method is based on: (a) enzymatic DNA digestion; (b) HPLC separation of the resultant nucleosides; (c) acetylation of the oxidized nucleosides with [ 3H]Ac 2O (acetic anhydride); (d) removal of the radioactive debris; and (e) quantitative analysis of tritiated nucleoside acetates by HPLC. Enzymatic DNA digestion was optimized using DNase I in the presence of Mg 2+ (pH 7), followed by nuclease P1 in the presence of Zn 2+ (pH 5.1) and alkaline phosphatase (pH 7.5). Analysis of DNA oxidized with H 2O 2 in the presence of Fe 2+/EDTA for 30 min showed that the levels of 8-OHdG (8-hydroxy-2′-deoxyguanosine) were increased 2.7-fold, HMdU (5-hydroxymethyl-2′-deoxyuridine) 3.15-fold, and FdU (5-formyl-2′-deoxyuridine) 2.5-fold. Although the (−)-isomer of cis-dTG ( cis-thymidine glycol) was enhanced 2.3 times, the (+)-isomer remained virtually unchanged. Analysis of DNA isolated from epidermal cells of mice treated in vivo with the tumor promoter TPA (12- O-tetradecanoylphorbol 13-acetate) showed 4.8-, 2.7-, and 8.7-fold increases in the levels of total cis-dTG, 8-OHdG, and HMdU, respectively, and of some unknown DNA oxidation products. These results prove applicability of the 3H-postlabeling method to the analysis of DNA (and potentially RNA) isolated from many sources, including animals and humans.