Mass Spectrometric Methodology for the Determination of Glyoxaldeoxyguanosine and O6-Hydroxyethyldeoxyguanosine DNA Adducts Produced by Nitrosamine Bident Carcinogens

Abstract

N-Nitrosodiethanolamine (NDELA) is a bident carcinogen that undergoes both P-450 mediated alpha-hydroxylation and beta-oxidation, leading ultimately to the formation of two prominent DNA adducts, glyoxaldeoxyguanosine (gdG) and O6-2-hydroxyethyldeoxyguanosine (OHEdG), in rat liver. HPLC coupled with electrospray ionization (ESI) and tandem mass spectrometry was used for both detection and quantification of gdG and OHEdG. The method, which is fast, sensitive, and unambiguous, is a significant improvement over the previous 32P-postlabeling methodology. A rapid procedure for the enzymatic hydrolysis of the DNA under acidic conditions preserved the integrity of the pH sensitive gdG adducts. Glyoxal and 3-nitroso-2-oxazolidinone generated gdG and OHEdG adducts, respectively, in calf thymus DNA (ct-DNA) in a concentration (range of 10(4)) dependent manner permitting optimization. Isotopomeric internal standards were prepared from the modified guanine derivatives by enzymatic trans-glycosylation. Quantitative HPLC-ESI-MS/MS analysis employing selective reaction monitoring (SRM) for the loss of the deoxyribose fragment was utilized. Both adducts could be detected in the liver DNA of rats that were administered NDELA in a dose range of 0.4-0.8 mmol/kg. At the highest dose, gdG adducts (4.4-11 adducts/10(6) nuc.) were more abundant than OHEdG adducts (0.35-0.87 adducts/10(6) nuc.). Conversely, OHEdG adducts were produced in higher yields in ct-DNA than were gdG adducts at the same reagent concentrations.