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Quantitative analysis of the oxidative DNA lesion, 2,2-diamino-4-(2-deoxy-β-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone (oxazolone), in vitro and in vivo by isotope dilution-capillary HPLC-ESI-MS/MS

Author:
Matter, Brock, Malejka-Giganti, Danuta, Csallany, A. Saari, Tretyakova, Natalia
Source:
Nucleic acids research 2006 v.34 no.19 pp. 5449-5460
ISSN:
0305-1048
Subject:
DNA, DNA damage, animal fats and oils, biomarkers, calves, diet, dose response, guanine, hydrolysates, ionization, irradiation, isotope dilution technique, isotopes, liver, oxidants, oxidative stress, photooxidation, quantitative analysis, rats, riboflavin, site-directed mutagenesis, tandem mass spectrometry, thymus gland
Abstract:
A major DNA oxidation product, 2,2-diamino-4-[(2-deoxy-β-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone (oxazolone), can be generated either directly by oxidation of dG or as a secondary oxidation product with an intermediate of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG). Site-specific mutagenesis studies indicate that oxazolone is a strongly mispairing lesion, inducing ~10-fold more mutations than 8-oxo-dG. While 8-oxo-dG undergoes facile further oxidation, oxazolone appears to be a stable final product of guanine oxidation, and, if formed in vivo, can potentially serve as a biomarker of DNA damage induced by oxidative stress. In this study, capillary liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) methods were developed to enable quantitative analysis of both 8-oxo-dG and oxazolone in DNA from biological sources. Sensitive and specific detection of 8-oxo-dG and oxazolone in enzymatic DNA hydrolysates was achieved by isotope dilution with the corresponding ¹⁵N-labeled internal standards. Both nucleobase adducts were formed in a dose-dependent manner in calf thymus DNA subjected to photooxidation in the presence of riboflavin. While the amounts of oxazolone continued to increase with the duration of irradiation, those of 8-oxo-dG reached a maximum at 20 min, suggesting that 8-oxo-dG is converted to secondary oxidation products. Both lesions were found in rat liver DNA isolated under carefully monitored conditions to minimize artifactual oxidation. Liver DNA of diabetic and control rats maintained on a diet high in animal fat contained 2-6 molecules of oxazolone per 10⁷ guanines, while 8-oxo-dG amounts in the same samples were between 3 and 8 adducts per 10⁶ guanines. The formation of oxazolone lesions in rat liver DNA, their relative stability in the presence of oxidants and their potent mispairing characteristics suggest that oxazolone may play a role in oxidative stress-mediated mutagenesis.
Agid:
1963696