7-(2'-Carboxyethyl)guanine (7-CEG) and N2-(1'-carboxyethyl)guanine (N2-CEG) are carboxyethyl DNA adducts originating from different sources. 7-CEG could potentially be formed upon the metabolism of the carcinogenic nitrosamines 3-(methylnitrosamino)propionic acid (MNPA) and 1-nitroso-5,6-dihydrouracil, or by reaction of DNA with acrylic acid. N2-CEG could be formed upon exposure to methylglyoxal (MG), a mutagenic compound which is a major breakdown product of carbohydrates and is present in many foods and in cigarette smoke. We have previously analyzed human liver DNA for 7-CEG. The method was now further developed to include the quantitation of N2-CEG. A standard for N2-CEG was synthesized from dG and 1,1-dihydroxyacetone, purified and characterized by NMR and MS. The accuracy and precision of this new quantitative LC-NSI-HRMS/MS method was determined for both adducts. Investigation of potential artefactual formation of the adducts was performed. Finally, samples from 20 smokers and 20 nonsmokers were analyzed. Leukocyte DNA was isolated and hydrolyzed enzymatically, the samples were purified by cation exchange/reverse phase solid-phase extraction, and the resulting fraction containing the adducts was treated with acetyl chloride in methanol to convert the adducts to their methyl esters. Further purification and enrichment was performed using polymeric reverse phase solid-phase extraction, with the final fraction containing both adducts analyzed by LC-NSI-HRMS/MS with extracted ion chromatogram generation of the m/z 238 [M+H]+→152.0565 [BH]+ and m/z 238 [M+H]+ →178.0723 [M+H -COOCH3]+ transitions respectively, with a mass tolerance of 5 ppm. The analysis resulted in levels of 7-CEG (mean ± SD) of 602 ± 245 fmol/µmol dG in smokers, and 555 ± 171 fmol/µmol dG in nonsmokers, and of N2-CEG of 39.5 ± 10.6 pmol/µmol dG in smokers, and 33.8 ± 8.0 pmol/µmol dG in nonsmokers. Although a significant difference between smokers and nonsmokers was not observed, the method described here is unique in the use of high resolution mass spectrometry and establishes for the first time the ability to simultaneously quantify these two carboxyethyl adducts in leukocyte DNA.

Quantitation of carboxyethylguanine adducts in leukocyte DNA from smokers and nonsmokers using LC-NSI-HRMS/MS

2019 Fall National Meeting

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