Identification of New Markers of Alcohol-Derived DNA Damage in Humans

Alcohol consumption is a risk factor for the development of several cancers, including those of the head and neck and the esophagus. The underlying mechanisms of alcohol-induced carcinogenesis remain unclear; however, at these sites, alcohol-derived acetaldehyde seems to play a major role. By reacting with DNA, acetaldehyde generates covalent modifications (adducts) that can lead to mutations. Previous studies have shown a dose dependence between levels of a major acetaldehyde-derived DNA adduct and alcohol exposure in oral-cell DNA. The goal of this study was to optimize a mass spectrometry (MS)-based DNA adductomic approach to screen for all acetaldehyde-derived DNA adducts to more comprehensively characterize the genotoxic effects of acetaldehyde in humans. A high-resolution/-accurate-mass data-dependent constant-neutral-loss-MS3 methodology was developed to profile acetaldehyde-DNA adducts in purified DNA. This resulted in the identification of 22 DNA adducts. In addition to the expected N2-ethyldeoxyguanosine (after NaBH3CN reduction), two previously unreported adducts showed prominent signals in the mass spectra. MSn fragmentation spectra and accurate mass were used to hypothesize the structure of the two new adducts, which were then identified as N6-ethyldeoxyadenosine and N4-ethyldeoxycytidine by comparison with synthesized standards. These adducts were quantified in DNA isolated from oral cells collected from volunteers exposed to alcohol, revealing a significant increase after the exposure. In addition, 17 of the adducts identified in vitro were detected in these samples confirming our ability to more comprehensively characterize the DNA damage deriving from alcohol exposures.

A fast screening method for tricyclic antidepressants and their urinary metabolites by FAB-tandem mass spectrometry

A screening method for tricyclic antidepressants (TCA) and their urinary metabolites by FAB tandem mass spectrometry (FAB MS/MS) is presented. The collision induced dissociation (CID) behavior of pure dibenzocycloheptadienes (amitriptyline1, nortriptyline2), dibenzoxepines (doxepine3, dosulepine4), dibenzazepines (imipramine5, desipramine6, trimipramine7) and the iminostilbene derivative opipramol8is described and used for the specific determination of these tricyclics by MS/MS.For the screening procedure a methanolic extract of the urine sample is passed through a XAD-2 column and the eluate is examined without further purification after evaporation of the solvent. Identification of the TCA is achieved by constant neutral loss (CNL) scans for 173 u, 195 u and 193 u and subsequent daughter ion spectra of the molecular ions [M+H]+. Glucuronide conjugate metabolites of the tricyclic antidepressants1,2,3,6,8could be identified by CNL-scans (176 u) and the assignments were confirmed by daughter ion mass spectra. A urine sample of a fatal1,2,8overdose and samples of three patients under therapeutic dosage with1,3or6are presented as examples for application.