Nucleic Acids Research Advance Access originally published online on October 4, 2006
Nucleic Acids Research 2006 34(19):5449-5460; doi:10.1093/nar/gkl596
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Nucleic Acids Research, 2006, Vol. 34, No. 19 5449-5460
© 2006 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Chemistry |
Quantitative analysis of the oxidative DNA lesion, 2,2-diamino-4-(2-deoxy-ß-D-erythro-pentofuranosyl)amino]-5(2H)-oxazolone (oxazolone), in vitro and in vivo by isotope dilution-capillary HPLC-ESI-MS/MS
Department of Medicinal Chemistry, University of Minnesota Cancer Center, University of Minnesota Minneapolis, MN 55455, USA 1 Veterans Affairs Medical Center, Minneapolis MN 55417, USA 2 Department of Laboratory Medicine and Pathology, University of Minnesota Minneapolis, MN 55455, USA 3 Department of Food Science and Nutrition, University of Minnesota St Paul, MN 55108, USA
*To whom correspondence should be addressed at 760E CCRB, University of Minnesota Cancer Center, 420 Delaware St SE, Mayo Mail Code 806, Minneapolis, MN 55455, USA; Tel: +1 612 626 3432; Fax +1 612 626 5135; Email: trety001{at}umn.edu
Received April 12, 2006. Revised July 7, 2006. Accepted August 1, 2006.
A major DNA oxidation product, 2,2-diamino-4-[(2-deoxy-ß-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.
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