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

Matter B, Malejka-Giganti D, Csallany AS, Tretyakova N - Nucleic Acids Res. (2006)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicinal Chemistry, University of Minnesota Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.

ABSTRACT
A major DNA oxidation product, 2,2-diamino-4-[(2-deoxy-beta-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 approximately 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 10(7) guanines, while 8-oxo-dG amounts in the same samples were between 3 and 8 adducts per 10(6) 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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Formation of 8-oxo-dG and oxazolone in calf thymus DNA subjected to photooxidation in the presence of riboflavin. Aliquots of 50 μl were withdrawn at 0, 5, 10, 15, 20, 30 and 60 min, and analyzed as shown in Scheme 2.
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fig4: Formation of 8-oxo-dG and oxazolone in calf thymus DNA subjected to photooxidation in the presence of riboflavin. Aliquots of 50 μl were withdrawn at 0, 5, 10, 15, 20, 30 and 60 min, and analyzed as shown in Scheme 2.

Mentions: The newly developed HPLC-ESI-MS/MS methods were tested by analyzing 8-oxo-dG and oxazolone in calf thymus DNA subjected to photooxidation in the presence of riboflavin. Riboflavin is an endogenous cellular photosensitizer that acts via the Type I mechanism, directly transferring a single electron from guanine molecule to the excited molecule of the dye (37). This initiates a series of reactions, ultimately leading to 8-oxo-dG and oxazolone (36,39). Under our experimental conditions (0°C, constant purging with air), amounts of oxazolone in DNA increased linearly with the duration of exposure to visible light (5–60 min irradiation, Figure 4). At short incubation times (0–20 min), 8-oxo-dG levels were comparable to those of oxazolone. When photooxidation time was extended to 30–60 min, oxazolone amounts continued to increase, suggesting that it was refractory to further oxidation in the presence of Type I photo-oxidizing agents (Figure 4). In contrast, 8-oxo-dG amounts reached a plateau following 20 min reaction, probably as a result of its oxidation to secondary products. Taken together with validation experiments described above, these in vitro studies confirmed that our HPLC-ESI-MS/MS methodology could be used for accurate and reliable quantification of both oxidative lesions in DNA hydrolysates from rat tissues.


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

Matter B, Malejka-Giganti D, Csallany AS, Tretyakova N - Nucleic Acids Res. (2006)

Formation of 8-oxo-dG and oxazolone in calf thymus DNA subjected to photooxidation in the presence of riboflavin. Aliquots of 50 μl were withdrawn at 0, 5, 10, 15, 20, 30 and 60 min, and analyzed as shown in Scheme 2.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1636462&req=5

fig4: Formation of 8-oxo-dG and oxazolone in calf thymus DNA subjected to photooxidation in the presence of riboflavin. Aliquots of 50 μl were withdrawn at 0, 5, 10, 15, 20, 30 and 60 min, and analyzed as shown in Scheme 2.
Mentions: The newly developed HPLC-ESI-MS/MS methods were tested by analyzing 8-oxo-dG and oxazolone in calf thymus DNA subjected to photooxidation in the presence of riboflavin. Riboflavin is an endogenous cellular photosensitizer that acts via the Type I mechanism, directly transferring a single electron from guanine molecule to the excited molecule of the dye (37). This initiates a series of reactions, ultimately leading to 8-oxo-dG and oxazolone (36,39). Under our experimental conditions (0°C, constant purging with air), amounts of oxazolone in DNA increased linearly with the duration of exposure to visible light (5–60 min irradiation, Figure 4). At short incubation times (0–20 min), 8-oxo-dG levels were comparable to those of oxazolone. When photooxidation time was extended to 30–60 min, oxazolone amounts continued to increase, suggesting that it was refractory to further oxidation in the presence of Type I photo-oxidizing agents (Figure 4). In contrast, 8-oxo-dG amounts reached a plateau following 20 min reaction, probably as a result of its oxidation to secondary products. Taken together with validation experiments described above, these in vitro studies confirmed that our HPLC-ESI-MS/MS methodology could be used for accurate and reliable quantification of both oxidative lesions in DNA hydrolysates from rat tissues.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicinal Chemistry, University of Minnesota Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA.

ABSTRACT
A major DNA oxidation product, 2,2-diamino-4-[(2-deoxy-beta-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 approximately 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 10(7) guanines, while 8-oxo-dG amounts in the same samples were between 3 and 8 adducts per 10(6) 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.

Show MeSH
Related in: MedlinePlus