Endemic (Balkan) nephropathy (EN),environmental mutagen ͉ p53 mutation ͉ urothelial cancer ͉ DNA adduct

No abstract

An accurate and sensitive liquid chromatography-electrospray ionization/multi-stage mass spectrometry (LC-ESI/MS/MS n ) technique has been developed for the characterization and quantification of 2′-deoxyguanosine (dG) adducts of the dietary mutagen, 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP). PhIP is an animal and potential human carcinogen that occurs in grilled meats. Following enzymatic digestion and adduct enrichment by solid-phase extraction (SPE), PhIP-DNA adducts were analyzed by MS/MS and MS n scan modes on a 2-D linear quadrupole ion trap mass spectrometer (QIT/MS). The major DNA adduct, N-(deoxyguanosin-8-yl)-2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (dG-C8-PhIP), was detected in calf thymus (CT) DNA modified in vitro with a bioactivated form of PhIP and in the colon and liver of rats given PhIP as part of the diet. The lower limit of detection (LOD) was 1 adduct per 10 8 DNA bases, and the limit of quantification (LOQ) was 3 adducts per 10 8 DNA bases in both MS/MS and MS 3 scan modes, using 27 μg of DNA for analysis. Measurements were based on isotope dilution with the internal standard, N-(deoxyguanosin-8-yl)-2-amino-1-(trideutero)methyl-6-phenylimidazo [4,5-b] pyridine (dG-C8-[ 2 H 3 C]-PhIP). The selected reaction monitoring (SRM) scan mode in MS/MS was employed to monitor the loss of deoxyribose (dR) from the protonated molecules of the adducts ([M + H -116] + ). The consecutive reaction monitoring (CRM) scan modes in MS 3 and MS 4 were used to measure and further characterize product ions of the aglycone ion (BH 2 + ) (Guanyl-PhIP). The MS 3 scan mode was effective in eliminating isobaric interferences observed in the MS/MS scan mode and resulted in an improved signal-to-noise (S/N) ratio. Moreover, the product ion spectra obtained by the MS n scan modes provided rich structural information about the adduct and were used to corroborate the identity of dG-C8-PhIP. In addition, an isomeric dG-PhIP adduct was detected in vivo. This LCESI/MS/MS n method is the first reported application on the use of the MS 3 scan mode for the analysis of DNA adducts in vivo.