Quantitative high-performance liquid chromatography analysis of DNA oxidized in vitro and in vivo.
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]Ac2O (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 Mg2+ (pH 7), followed by nuclease P1 in the presence of Zn2+ (pH 5.1) and alkaline phosphatase (pH 7.5). Analysis of DNA oxidized with H2O2 in the presence of Fe2+/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.
Frenkel
K
K
Department of Environmental Medicine, New York University Medical Center, New York 10016.
Zhong
Z J
ZJ
Wei
H C
HC
Karkoszka
J
J
Patel
U
U
Rashid
K
K
Georgescu
M
M
Solomon
J J
JJ
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis / Q000378:metabolism; D004926:Escherichia coli; D051379:Mice; D015394:Molecular Structure; D009705:Nucleosides / Q000378:metabolism; D010084:Oxidation-Reduction; D013755:Tetradecanoylphorbol Acetate
D004269:DNA, Bacterial; D009705:Nucleosides; D004247:DNA; D013755:Tetradecanoylphorbol Acetate