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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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HPLC-ESI+-MS/MS analysis of 8-oxo-dG in an enzymatic hydrolysate of rat liver DNA (80 μg). An Agilent 1100 series capillary HPLC-ion trap MS system was used. A Zorbax SB C18 column (0.5 × 150 mm, 5 (m) was maintained at 10°C and eluted at a flow rate of 12 (l/min with a gradient of methanol (solvent B) in 15 mM ammonium acetate (solvent A). The mass spectrometer was operated in the positive ion MS/MS mode. Quantitative analyses were performed in selected reaction monitoring mode using the transitions m/z 284.1→168.0 (M + 2H − dR)+ for 8-oxo-dG and the corresponding transition m/z 289.1→173 for [15N5]-8-oxo-dG.
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fig6: HPLC-ESI+-MS/MS analysis of 8-oxo-dG in an enzymatic hydrolysate of rat liver DNA (80 μg). An Agilent 1100 series capillary HPLC-ion trap MS system was used. A Zorbax SB C18 column (0.5 × 150 mm, 5 (m) was maintained at 10°C and eluted at a flow rate of 12 (l/min with a gradient of methanol (solvent B) in 15 mM ammonium acetate (solvent A). The mass spectrometer was operated in the positive ion MS/MS mode. Quantitative analyses were performed in selected reaction monitoring mode using the transitions m/z 284.1→168.0 (M + 2H − dR)+ for 8-oxo-dG and the corresponding transition m/z 289.1→173 for [15N5]-8-oxo-dG.

Mentions: The formation of 8-oxo-dG and oxazolone in DNA isolated from livers of control and diabetic rats maintained on a high fat, beef tallow-based diet was examined. A previous study (45) revealed that diabetic rats excreted increased amounts of lipophilic aldehydes and related carbonyl compounds in their urine as an indication of elevated lipid peroxidation. Representative HPLC-ESI-MS/MS traces for analyses of 8-oxo-dG and oxazolone in rat liver samples are shown in Figures 5 and 6, respectively. Oxazolone amounts in rat liver DNA (2–6 adducts per 107 normal guanines) were approximately an order of magnitude lower than those of 8-oxo-dG in the same tissue (3–8 adducts per 106 G) (Table 1). The levels of 8-oxo-dG detected in our study were similar to those determined by a similar method in the livers of control Sprague–Dawley rats (54). Streptozotocin-induced diabetic rats contained slightly higher levels of 8-oxo-dG than control animals (P = 0.009, Table 1), which was consistent with earlier reports of diabetes-induced increase in urinary 8-hydroxy-2′-deoxyguanosine (55). However, no significant differences were observed between oxazolone levels in liver DNA extracted from the two groups of animals (P = 0.5, Table 1).


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)

HPLC-ESI+-MS/MS analysis of 8-oxo-dG in an enzymatic hydrolysate of rat liver DNA (80 μg). An Agilent 1100 series capillary HPLC-ion trap MS system was used. A Zorbax SB C18 column (0.5 × 150 mm, 5 (m) was maintained at 10°C and eluted at a flow rate of 12 (l/min with a gradient of methanol (solvent B) in 15 mM ammonium acetate (solvent A). The mass spectrometer was operated in the positive ion MS/MS mode. Quantitative analyses were performed in selected reaction monitoring mode using the transitions m/z 284.1→168.0 (M + 2H − dR)+ for 8-oxo-dG and the corresponding transition m/z 289.1→173 for [15N5]-8-oxo-dG.
© Copyright Policy
Related In: Results  -  Collection

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

fig6: HPLC-ESI+-MS/MS analysis of 8-oxo-dG in an enzymatic hydrolysate of rat liver DNA (80 μg). An Agilent 1100 series capillary HPLC-ion trap MS system was used. A Zorbax SB C18 column (0.5 × 150 mm, 5 (m) was maintained at 10°C and eluted at a flow rate of 12 (l/min with a gradient of methanol (solvent B) in 15 mM ammonium acetate (solvent A). The mass spectrometer was operated in the positive ion MS/MS mode. Quantitative analyses were performed in selected reaction monitoring mode using the transitions m/z 284.1→168.0 (M + 2H − dR)+ for 8-oxo-dG and the corresponding transition m/z 289.1→173 for [15N5]-8-oxo-dG.
Mentions: The formation of 8-oxo-dG and oxazolone in DNA isolated from livers of control and diabetic rats maintained on a high fat, beef tallow-based diet was examined. A previous study (45) revealed that diabetic rats excreted increased amounts of lipophilic aldehydes and related carbonyl compounds in their urine as an indication of elevated lipid peroxidation. Representative HPLC-ESI-MS/MS traces for analyses of 8-oxo-dG and oxazolone in rat liver samples are shown in Figures 5 and 6, respectively. Oxazolone amounts in rat liver DNA (2–6 adducts per 107 normal guanines) were approximately an order of magnitude lower than those of 8-oxo-dG in the same tissue (3–8 adducts per 106 G) (Table 1). The levels of 8-oxo-dG detected in our study were similar to those determined by a similar method in the livers of control Sprague–Dawley rats (54). Streptozotocin-induced diabetic rats contained slightly higher levels of 8-oxo-dG than control animals (P = 0.009, Table 1), which was consistent with earlier reports of diabetes-induced increase in urinary 8-hydroxy-2′-deoxyguanosine (55). However, no significant differences were observed between oxazolone levels in liver DNA extracted from the two groups of animals (P = 0.5, Table 1).

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