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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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Experimental scheme for HPLC-ESI-MS/MS analysis of 8-oxo-dG and oxazolone in DNA.
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sch2: Experimental scheme for HPLC-ESI-MS/MS analysis of 8-oxo-dG and oxazolone in DNA.

Mentions: Oxazolone analysis was performed with a Thermo-Finnigan TSQ Quantum Ultra mass spectrometer interfaced with an Agilent 1100 capillary HPLC. A Thermo Hypersil-Keystone (Bellefonte, PA) Hypercarb column (0.5 × 100 mm, 5 μm) was eluted with a gradient of isopropanol/acetonitrile (3:1) (solvent B) in 0.05% acetic acid (solvent A) (0 min, 0% B; 1 min, 0% B; 8.5 min, 10.7% B; 9.3 min, 0% B; 20 min, 0% B) at a flow rate of 12 μl/min. The mass spectrometer was operated in the ESI+ MS/MS mode. The spray voltage was set to 3.1 kV, the source temperature was 250°C, and the sheath gas pressure was 30 psi. Product ions of m/z 247.1 ([M + H]+ of oxazolone nucleoside) and m/z 251.1 (15N4-oxazolone, internal standard) were obtained, with a collision energy of 14 and a collision gas pressure of 1 mtorr. The peak width for Q1 and Q3 were both set to 0.70 amu. Quantitative analyses were performed in selected reaction monitoring mode using the transitions m/z 247.1→87.1 [M + 2H − dR − CO2]+ and m/z 251.1→91.1 for oxazolone and 15N4-oxazolone, respectively. The method was validated by spiking calf thymus DNA (0.2 mg) with 2 pmol each oxazolone and 15N4-oxazolone, followed by enzymatic hydrolysis, HPLC cleanup and HPLC-ESI+-MS/MS analysis as described above (Scheme 2). Spiked samples were quantified to contain 96.7 ± 1.3% of the target oxazolone amount (n = 5). The same experiment performed in the absence of DNA (pure standards) yielded an accuracy of 100–103%, with a precision of 2.62% (N = 8, during several days). The lower limit of detection for oxazolone standard was estimated as 5 fmol on column (S/N = 4).


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)

Experimental scheme for HPLC-ESI-MS/MS analysis of 8-oxo-dG and oxazolone in DNA.
© Copyright Policy
Related In: Results  -  Collection

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

sch2: Experimental scheme for HPLC-ESI-MS/MS analysis of 8-oxo-dG and oxazolone in DNA.
Mentions: Oxazolone analysis was performed with a Thermo-Finnigan TSQ Quantum Ultra mass spectrometer interfaced with an Agilent 1100 capillary HPLC. A Thermo Hypersil-Keystone (Bellefonte, PA) Hypercarb column (0.5 × 100 mm, 5 μm) was eluted with a gradient of isopropanol/acetonitrile (3:1) (solvent B) in 0.05% acetic acid (solvent A) (0 min, 0% B; 1 min, 0% B; 8.5 min, 10.7% B; 9.3 min, 0% B; 20 min, 0% B) at a flow rate of 12 μl/min. The mass spectrometer was operated in the ESI+ MS/MS mode. The spray voltage was set to 3.1 kV, the source temperature was 250°C, and the sheath gas pressure was 30 psi. Product ions of m/z 247.1 ([M + H]+ of oxazolone nucleoside) and m/z 251.1 (15N4-oxazolone, internal standard) were obtained, with a collision energy of 14 and a collision gas pressure of 1 mtorr. The peak width for Q1 and Q3 were both set to 0.70 amu. Quantitative analyses were performed in selected reaction monitoring mode using the transitions m/z 247.1→87.1 [M + 2H − dR − CO2]+ and m/z 251.1→91.1 for oxazolone and 15N4-oxazolone, respectively. The method was validated by spiking calf thymus DNA (0.2 mg) with 2 pmol each oxazolone and 15N4-oxazolone, followed by enzymatic hydrolysis, HPLC cleanup and HPLC-ESI+-MS/MS analysis as described above (Scheme 2). Spiked samples were quantified to contain 96.7 ± 1.3% of the target oxazolone amount (n = 5). The same experiment performed in the absence of DNA (pure standards) yielded an accuracy of 100–103%, with a precision of 2.62% (N = 8, during several days). The lower limit of detection for oxazolone standard was estimated as 5 fmol on column (S/N = 4).

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