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

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.

Measurement and Meaning of Oxidatively Modified DNA Lesions in Urine

“…By the same token, oxidation of Thy to thymine glycol is less likely to occur than Gua to 8-oxoGua due to the lower oxidation potential of Gua compared with Thy. However, it would seem that most of these studies have examined the oxidation of 8-oxoGua in situ in DNA (84), rather than as a postexcision product of DNA repair, which may alter the likelihood of oxidation and its ''oxidizability.'' In contrast, M 1 G and 3-(2-deoxy-h-D-erythro-pentofuranosyl)pyrimido[1,2-a]purin-10(3H)-one both seem to undergo further oxidative metabolism in rat liver cytosol, with the base adduct being a better substrate for such enzymic oxidation than the deoxyribonucleoside adduct (85,86).…”

DNA Base Damage by Reactive Oxygen Species, Oxidizing Agents, and UV Radiation

“…This may be accounted for by the lower oxygen concentration and the presence of reducing compounds such as thiols in the cellular environments. The formation of 2,2,4-triamino-5(2H )-oxazolone (oxazolone), a welldocumented † OH and one-electron oxidation product of Gua nucleoside , has been detected in the hepatic DNA of diabetic rats, albeit the yield was tenfold lower than that of 8-oxoGua (Matter et al 2006). The formation of oxazolone is rationalized in terms of initial † OH-mediated H-atom abstraction from the 2-amino group of guanine (Chatgilialoglu et al 2011a) as a more relevant alternative to † OH addition at C4 followed by dehydration, which was initially proposed several years ago (Candeias and Steenken 2000).…”

Synthesis and Characterization of Adducts Formed in the Reactions of Safrole 2′,3′‐Oxide with 2′‐Deoxyadenosine, Adenine, and Calf Thymus DNA

“…A solution of DNA (100 μL, 100 μg) was hydrolyzed with a mixture of DNase I (4 U), phosphodiesterase I (32 mU), phosphodiesterase II (80 mU), and acid phosphatase (1 U) and incubated at 37°C for 8-10 h. [37,38] The amounts of reagents were adjusted according to the amount of DNA in the sample. To evaluate the efficiency of the enzymatic hydrolysis, calibration curves of dAdo, dGuo, dCyd, and dThd were established by HPLC analysis.…”