Quantification of N2-Carboxymethyl-2′-deoxyguanosine in Calf Thymus DNA and Cultured Human Kidney Epithelial Cells by Capillary High-Performance Liquid Chromatography−Tandem Mass Spectrometry Coupled with Stable Isotope Dilution Method

Abstract

Glyoxal is generated endogenously from the degradation of glucose and the oxidation of carbohydrates, lipids, and the 2-deoxyribose moieties of DNA. Glyoxal is also widely used in industry and is present in cigarette smoke and food. Glyoxal can conjugate with nucleobases and proteins to give advanced glycation end products. N(2)-Carboxymethyl-2'-deoxyguanosine (N(2)-CMdG) and the cyclic 1,N(2)-glyoxal-dG are the major glyoxal adducts formed in DNA. In this study, we first assessed the stabilities of these two adducts. It turned out that 1,N(2)-glyoxal-dG was very unstable, with more than 70% of the adduct being decomposed to dG upon a 24 h incubation at 37 degrees C in phosphate-buffered saline. However, N(2)-CMdG was very stable; less than 0.5% of the lesion was degraded to dG after a 7 day incubation under the same conditions. We further developed a sensitive capillary liquid chromatography-electrospray ionization-tandem mass spectrometry coupled with a stable isotope dilution method and quantified the formation of N(2)-CMdG in calf thymus DNA and 293T human kidney epithelial cells that were exposed to glyoxal and in calf thymus DNA treated with d-glucose. Our results showed that N(2)-CMdG was produced at 2-134 lesions per 10(6) nucleosides in calf thymus DNA when the surrounding glyoxal concentration was increased from 10 to 500 microM and approximately 3-27 lesions per 10(7) nucleosides, while the D-glucose concentration changed from 2 to 50 mM. Furthermore, N(2)-CMdG was induced endogenously in 293T human kidney epithelial cells and exposure to glyoxal further stimulated the formation of this lesion; the level of this adduct ranged from 7 to 15 lesions per 10(8) nucleosides, while the glyoxal concentration increased from 10 microM to 1.25 mM. Collectively, our results suggested that N(2)-CMdG might serve as a biomarker for glyoxal exposure.