The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, 1) is a potent lung carcinogen in laboratory animals and is believed to play a key role in the development of lung cancer in smokers. Metabolic activation of NNK leads to the formation of pyridyloxobutyl DNA adducts, a critical step in its mechanism of carcinogenesis. In addition to DNA nucleobase adducts, DNA phosphate adducts can be formed by pyridyloxobutylation of the oxygen atoms of the internucleotidic phosphodiester linkages. We report the use of a liquid chromatography–nanoelectrospray ionization–high-resolution tandem mass spectrometry technique to characterize 30 novel pyridyloxobutyl DNA phosphate adducts in calf thymus DNA (CT-DNA) treated with 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanone (NNKOAc, 2), a regiochemically activated form of NNK. A 15N3-labeled internal standard was synthesized for one of the most abundant phosphate adducts, dCp[4-oxo-4-(3-pyridyl)butyl]dC (CpopC), and this standard was used to quantify CpopC and to estimate the levels of other adducts in the NNKOAc-treated CT-DNA. Formation of DNA phosphate adducts by NNK in vivo was further investigated in rats treated with NNK acutely (0.1 mmol/kg once daily for 4 days by subcutaneous injection) and chronically (5 ppm in drinking water for 10, 30, 50, and 70 weeks). This study provides the first comprehensive structural identification and quantitation of a panel of DNA phosphate adducts of a structurally complex carcinogen and chemical support for future mechanistic studies of tobacco carcinogenesis in humans.

Regulated transport and local translation of mRNA in neurons are critical for modulating synaptic strength, maintaining proper neural circuitry, and establishing long term memory. Neuronal RNA granules are ribonucleoprotein particles that serve to transport mRNA along microtubules and control local protein synthesis in response to synaptic activity. Studies suggest that neuronal RNA granules share similar structures and functions with somatic P-bodies. We recently reported that the Huntington disease protein huntingtin (Htt) associates with Argonaute (Ago) and localizes to cytoplasmic P-bodies, which serve as sites of mRNA storage, degradation, and small RNAmediated gene silencing. Here we report that wild-type Htt associates with Ago2 and components of neuronal granules and cotraffics with mRNA in dendrites. Htt was found to co-localize with RNA containing the 3-untranslated region sequence of known dendritically targeted mRNAs. Knockdown of Htt in neurons caused altered localization of mRNA. When tethered to a reporter construct, Htt down-regulated reporter gene expression in a manner dependent on Ago2, suggesting that Htt may function to repress translation of mRNAs during transport in neuronal granules.

Quantitation of DNA adducts could provide critical information on the relationship between exposure to tobacco smoke and cancer risk in smokers. In this study, we developed a robust and sensitive liquid chromatography-tandem mass spectrometry method for the analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB1)-releasing DNA adducts in human oral cells, a non-invasive source of DNA for biomarker studies. Isolated DNA undergoes acid hydrolysis, after which samples are purified by solid-phase extraction and analyzed by LC-ESI-MS/MS. The developed method was applied for analysis of samples obtained via collection with a commercial mouthwash from 30 smokers and 15 nonsmokers. In smokers, the levels of HPB-releasing DNA adducts averaged 12.0 pmol HPB/mg DNA (detected in 20 out of 28 samples with quantifiable DNA yield) and in nonsmokers, the levels of adducts averaged 0.23 pmol/mg DNA (detected in 3 out of 15 samples). For the 30 smoking subjects, matching buccal brushings were also analyzed and HPB-releasing DNA adducts were detected in 24 out of 27 samples with quantifiable DNA yield, averaging 44.7 pmol HPB/mg DNA. The levels of adducts in buccal brushings correlated with those in mouthwash samples of smokers (R = 0.73, p < 0.0001). Potentially the method can be applied in studies of individual susceptibility to tobacco-induced cancers in humans.

Many harmful constituents are present in e-cigarettes at much lower levels than in cigarette smoke, and the results of analysis of urinary biomarkers in e-cigarette users are consistent with these findings. However, understanding the health effects of chronic exposures to e-cigarette aerosols may require thinking beyond these comparisons. In this study, we investigated the endogenous formation of the tobacco-specific oral and esophageal carcinogen N'-nitrosonornicotine (NNN) in e-cigarette users. Salivary NNN, nornicotine, and nicotine as well as urinary tobacco biomarkers, including total NNN, were analyzed in 20 e-cigarette users, 20 smokers, and 19 nonsmokers. Nornicotine and NNN levels in e-cigarettes used by the study participants were also analyzed. The mean of NNN in saliva of e-cigarette users was 14.6 (±23.1) pg/mL, ranging from nonquantifiable (below the limit of quantitation, LOQ) to 76.0 pg/mL. In smokers, salivary NNN ranged from below LOQ to 739 pg/mL, with 80% of smokers having salivary NNN in the range of levels found in e-cigarette users. Consistent with a previous report, very low levels of urinary total NNN were present in only 5 out of 20 e-cigarette users (ranging from 0.001 to 0.01 pmol/mL urine). Only trace levels of NNN were found in e-cigarette liquids. Together, our findings demonstrate that NNN is formed endogenously in e-cigarette users. While the overall exposure to NNN in e-cigarette users is dramatically lower than in smokers, the known carcinogenic potency of NNN warrants further investigations into the potential consequences of its endogenous formation. Salivary NNN, rather than urinary total NNN, which accounts for only 1-3% of the NNN dose, should be used to monitor e-cigarette users' exposure to this carcinogen.

Transport of mRNAs to diverse neuronal locations via RNA granules serves an important function in regulating protein synthesis within restricted sub-cellular domains. We recently detected the Huntington's disease protein huntingtin (Htt) in dendritic RNA granules; however, the functional significance of this localization is not known. Here we report that Htt and the huntingtin-associated protein 1 (HAP1) are co-localized with the microtubule motor proteins, the KIF5A kinesin and dynein, during dendritic transport of β-actin mRNA. Live cell imaging demonstrated that β-actin mRNA is associated with Htt, HAP1, and dynein intermediate chain in cultured neurons. Reduction in the levels of Htt, HAP1, KIF5A, and dynein heavy chain by lentiviral-based shRNAs resulted in a reduction in the transport of β-actin mRNA. These findings support a role for Htt in participating in the mRNA transport machinery that also contains HAP1, KIF5A, and dynein.

DNA adducts are believed to play a central role in the induction of cancer in cigarette smokers and are proposed as being potential biomarkers of cancer risk. We have summarized research conducted since 2012 on DNA adduct formation in smokers. A variety of DNA adducts derived from various classes of carcinogens, including aromatic amines, polycyclic aromatic hydrocarbons, tobacco-specific nitrosamines, alkylating agents, aldehydes, volatile carcinogens, as well as oxidative damage have been reported. The results are discussed with particular attention to the analytical methods used in those studies. Mass spectrometry-based methods that have higher selectivity and specificity compared to 32P-postlabeling or immunochemical approaches are preferred. Multiple DNA adducts specific to tobacco constituents have also been characterized for the first time in vitro or detected in vivo since 2012, and descriptions of those adducts are included. We also discuss common issues related to measuring DNA adducts in humans, including the development and validation of analytical methods and prevention of artifact formation.

SummaryThe blood and lymphatic vascular system of the gut plays an important role in tissue fluid homeostasis, nutrient absorption and immune surveillance. To obtain a better understanding of the anatomic basis of these functions, the blood and lymphatic vasculature of the lower segment of mouse gut and several constituents of gut‐associated lymphoid tissue (GALT) including Peyer's patch, specialized lymphoid nodules in the caecum, small lymphoid aggregates and lymphoid nodules in the colon were studied by using confocal microscopy. Additionally, the innervation and nerve/immune cell interactions in the gut and Peyer's patch were investigated by using cell surface marker PGP9.5 and Glial fibrillary acidic protein (GFAP). In the gut and Peyer's patch, the nerves have contact with B cell, T cell and B220CD3 double‐positive cells. Dendritic cells, the most important antigen‐presenting cells, were closely apposed to some nerves. Some dendritic cells formed membrane–membrane contact with nerve terminals and neuron cell body. Many fine nerve fibres, which are indirectly detected by GFAP, have contact with dendritic cells and other immune cells in the Peyer's patch. Furthermore, the expression of Muscarinic Acetylcholine receptor (subtype M2) was characterized on dendritic cells and other cell population. These findings are expected to provide a route to understand the anatomic basis of neuron‐immune regulation/cross‐talk and probably neuroinvasion of prion pathogens in the gut and GALT.

The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a powerful lung carcinogen in animal models and is considered a causative factor for lung cancer in people who use tobacco products. NNK undergoes metabolic activation-a critical step in its mechanism of carcinogenesis-to an intermediate which reacts with DNA to form pyridyloxobutyl DNA base and phosphate adducts. Another important metabolic pathway of NNK is its conversion to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which similarly forms pyridylhydroxybutyl DNA base adducts that have been characterized previously. In this study, we investigated the potential formation of pyridylhydroxybutyl DNA phosphate adducts. We report the characterization and quantitation of 107 structurally unique pyridylhydroxybutyl DNA phosphate adducts in the lungs of rats treated chronically with a carcinogenic dose of 5 ppm of NNK in their drinking water for up to 70 weeks, by using a novel liquid chromatography-nanoelectrospray ionization-high-resolution tandem mass spectrometry method. Our findings demonstrate that pyridylhydroxybutyl phosphate adducts account for 38-55 and 34-40% of all the measured pyridine-containing DNA adducts in rat lung and liver, respectively, upon treatment with NNK. Some of the pyridylhydroxybutyl DNA phosphate adducts persisted in both tissues for over 70 weeks, suggesting that they could be potential biomarkers of chronic exposure to NNK and NNAL. This study provides comprehensive characterization and relative quantitation of a panel of NNK/NNAL-derived DNA phosphate adducts, thus identifying NNK as the source of the most structurally diverse set of DNA adducts identified to date from any carcinogen.