Dataset Information

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.

SUBMITTER: Wang H

PROVIDER: S-EPMC2818644 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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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.

Wang Hongxia H Cao Huachuan H Wang Yinsheng Y

Chemical research in toxicology 20100101 1

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 stabilitie ...[more]

PMID: 19968260

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Project description:1. Rat thymus cells were incubated in homologous serum (10%) and medium 199. The effects of various quantities of thymidine or deoxycytidine (0-30mum) on the radioactive labelling of cells with the corresponding radioactive deoxynucleoside were examined. From plots of the reciprocal of the radioactivity incorporated against the total deoxynucleoside concentration (;isotope-dilution plots'), values were obtained for (a) the V(max.) of the rate-limiting step governing incorporation of the deoxynucleoside, and (b) the concentration of the pool of compounds competing with the radioactive deoxynucleoside at that rate-limiting step. From changes in these values under different experimental conditions inferences were drawn on the position and control of the rate-limiting step within intact cells. 2. Isotope-dilution plots for deoxycytidine were linear, whereas plots for thymidine were bimodal, indicating an abrupt increase in both the V(max.) and pool concentration at a critical thymidine concentration (approx. 5mum). The bimodality was removed by amethopterin. The V(max.) determined with deoxy[U-(14)C]cytidine was approximately equal to the sum of the V(max.) determined with deoxy[5-(3)H]cytidine and the V(max.) determined with [Me-(3)H]thymidine at thymidine concentrations above 5mum. 3. The thymidine competitor pool at thymidine concentrations above 5mum was approximately equal to the sum of the deoxycytidine competitor pool and the thymidine competitor pool at thymidine concentrations below 5mum. The pools were independent of cell concentration and dependent on serum concentration. 4. These results were explained on the following basis. Deoxycytidine in serum (16mum) is the major source of both cytosine and, by way of thymidylate synthetase, thymine, in the DNA of thymus cells. Serum deoxycytidine normally maintains a sufficient intracellular concentration of dTTP to inhibit partially the activity of thymidine kinase. When the dTTP concentration is lowered, either by decreasing the concentration of deoxycytidine or by inhibiting thymidylate synthetase, the activity of thymidine kinase increases. The activity of thymidine kinase may also be increased by concentrations of thymidine greater than 5mum, which overcome the inhibition of the enzyme by dTTP. At concentrations of thymidine below 5mum, thymidine kinase limits the rate of labelling with [Me-(3)H]thymidine and the radioactivity is diluted by a pool of unlabelled thymidine in serum (4mum). At thymidine concentrations greater than 5mum, the activity of DNA polymerase limits the rate of labelling and the radioactivity is diluted both by serum thymidine and, indirectly, by serum deoxycytidine.

Dataset Information

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.

Publications

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.

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