Cigarette smoking is an important source of human exposure to toxicants and carcinogens and contributes significantly to cancer morbidity and mortality worldwide. Acrolein, a widespread environmental pollutant, is present in relatively high amounts in cigarette smoke and can react directly with DNA to form DNA adducts, which serve as important biomarkers for the assessment of exposure to acrolein and its potential role in smoking related cancer. Etheno-DNA adducts are promutagenic DNA lesions that can derive from exogenous chemicals as well as endogenous sources, including lipid peroxidation. In this study, we developed a combined method for the quantitation of (6R/S)-3-(2′-deoxyribos-1′-yl)-5,6,7,8,-tetrahydro-6-hydroxypyrimido­[1,2-a]­purine-10­(3H)-one (α-OH-Acr-dGuo), (8R/S)-3-(2′-deoxyribos-1′-yl)-5,6,7,8,-tetrahydro-8-hydroxypyrimido­[1,2-a]­purine-10­(3H)-one (γ-OH-Acr-dGuo), 1,N 6-etheno-dAdo (εdAdo), and 3,N 4-etheno-dCyd (εdCyd) adducts in oral rinse and cytobrush DNA from smokers and nonsmokers by liquid chromatography–nanoelelctrospray ionization–high-resolution tandem mass spectrometry (LC-NSI-HRMS/MS). For oral rinse samples, there was a statistically significant difference between the levels of α-OH-Acr-dGuo, γ-OH-Acr-dGuo, εdAdo, and εdCyd in smokers (12.1 ± 17.9, 163 ± 227, 182 ± 568, and 194 ± 400 adducts/109 nucleotides, respectively) and nonsmokers (1.85 ± 2.08, 5.95 ± 4.23, 7.69 ± 11.7, and 6.07 ± 10.9 adducts/109 nucleotides, respectively). For cytobrush samples, there was a statistically significant difference between the levels of γ-OH-Acr-dGuo and εdAdo in smokers (259 ± 540 and 82.9 ± 271 adducts/109 nucleotides, respectively) and nonsmokers (7.37 ± 5.09 and 16.2 ± 30.2 adducts/109 nucleotides, respectively) but not for α-OH-Acr-dGuo and εdCyd. Our results demonstrate that oral mucosa cells are an excellent source of material for evaluating DNA adducts to be used as biomarkers of tobacco smoke exposure and molecular changes potentially related to cancer.

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.

An outbreak of cutaneous staphylococcosis caused high mortality among newborn and very young rabbits. The most typical lesions were exudative dermatitis in the youngest, subcutaneous abscesses in rabbits of all ages and mastitis in lactating does. Generalised staphylococcosis was a frequent secondary manifestation of the disease.

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), is a potent lung carcinogen that exerts its carcinogenic effects upon metabolic activation. The identification and quantitation of NNK metabolites could identify potential biomarkers of bioactivation and detoxification of this potent carcinogen and may be used to predict lung cancer susceptibility among smokers. Here, we used in vivo isotope-labeling and high-resolution-mass-spectrometry-based methods for the comprehensive profiling of all known and unknown NNK metabolites. The sample-enrichment, LC-MS, and data-analysis workflow, including a custom script for automated d- d- m/ z-pair-peak detection, enabled unbiased identification of numerous NNK metabolites. The structures of the metabolites were confirmed using targeted LC-MS with retention-time ( t) and MS-fragmentation comparisons to those of standards when possible. Eleven known metabolites and unchanged NNK were identified simultaneously. More importantly, our workflow revealed novel NNK metabolites, including 1,3-Diol (13), α-OH-methyl-NNAL-Gluc (14), nitro-NK- N-oxide (15), nitro-NAL- N-oxide (16), γ-OH NNAL (17), and three N-acetylcysteine (NAC) metabolites (18a-c). We measured the differences in the relative distributions of a panel of nitroso-containing NNK-specific metabolites in rats before and after phenobarbital (PB) treatment, and this served as a demonstration of a general strategy for the detection of metabolic differences in animal and cell systems. Lastly, we generated a d-labeled NNK-metabolite mixture to be used as internal standards ( d-rat urine) for the relative quantitation of NNK metabolites in humans, and this new strategy will be used to assess carcinogen exposure and ultimately to evaluate lung-cancer risk and susceptibility in smokers.

Reactive carbonyl compounds (RCCs) are ubiquitous in the environment and are generated endogenously as a result of various physiological and pathological processes. These compounds can react with biological molecules inducing deleterious processes believed to be at the basis of their toxic effects. Several of these compounds are considered to be implicated in neurotoxic processes, aging disorders, and cancer. Therefore, a method characterizing exposures to these chemicals will provide insights into how they may influence overall health and contribute to disease pathogenesis. Here, we have developed a high resolution accurate mass (HRAM) screening strategy allowing simultaneous identification and relative quantitation of DNPH-derivatized carbonyls in human biological fluids. The screening strategy involves the diagnostic neutral loss of hydroxyl radical triggering MS3 fragmentation, which is only observed in positive ionization mode of DNPH-derivatized carbonyls. Unique fragmentation pathways were used to develop a classification scheme for characterizing known and unanticipated/unknown carbonyl compounds present in saliva. Furthermore, a relative quantitation strategy was implemented to assess variations in the levels of carbonyl compounds before and after exposure using deuterated d3-DNPH. This relative quantitation method was tested on human samples before and after exposure to specific amounts of alcohol. The nano-electrospray ionization (nano-ESI) in positive mode afforded excellent sensitivity with detection limits on-column in the high-attomole levels. To the best of our knowledge, this is the first report of a method using HRAM neutral loss screening of carbonyl compounds. In addition, the method allows simultaneous characterization and relative quantitation of DNPH-derivatized compounds using nano-ESI in positive mode.