Composition of e-cigarette aerosols: A review and risk assessment of selected compounds.

The potential harms and benefits of e-cigarettes, or electronic nicotine delivery systems (ENDS), have received significant attention from public health and regulatory communities. Such products may provide a reduced risk means of nicotine delivery for combustible cigarette smokers while being inappropriately appealing to nicotine naive youth. Numerous authors have examined the chemical complexity of aerosols from various open- and closed-system ENDS. This body of literature is reviewed here, with the risks of ENDS aerosol exposure among users evaluated with a margin of exposure (MoE) approach for two non-carcinogens (methylglyoxal, butyraldehyde) and a cancer risk analysis for the carcinogen N-nitrosonornicotine (NNN). We identified 96 relevant papers, including 17, 13, and 5 reporting data for methylglyoxal, butyraldehyde, and NNN, respectively. Using low-end (minimum aerosol concentration, low ENDS use) and high-end (maximum aerosol concentration, high ENDS use) assumptions, estimated doses for methylglyoxal (1.78 × 10-3-135 µg/kg-bw/day) and butyraldehyde (1.9 × 10-4-66.54 µg/kg-bw/day) corresponded to MoEs of 227-17,200,000 and 271-280,000,000, respectively, using identified points of departure (PoDs). Doses of 9.90 × 10-6-1.99 × 10-4 µg/kg-bw/day NNN corresponded to 1.4-28 surplus cancers per 100,000 ENDS users, relative to a NNN-attributable surplus of 7440 per 100,000 cigarette smokers. It was concluded that methylglyoxal and butyraldehyde in ENDS aerosols, while not innocuous, did not present a significant risk of irritant effects among ENDS users. The carcinogenic risks of NNN in ENDS aerosols were reduced, but not eliminated, relative to concentrations reported in combustible cigarette smoke.

Pharmacological inhibition of AIMP2 aggregation attenuates α-synuclein aggregation and toxicity in Parkinson's disease.

The aggregation of aminoacyl transfer RNA synthetase complex-interacting multifunctional protein-2 (AIMP2) accelerates α-synuclein aggregation via direct interaction, leading to enhanced dopaminergic neurotoxicity in Parkinson's disease (PD). Thus, it would be beneficial to prevent AIMP2 aggregation to suppress α-synucleinopathy in PD. In this study, we screened small compounds that could inhibit the in vitro aggregation of AIMP2 using a 1909 small-compound library. The AIMP2 inhibitors (SAI-04, 06, and 08) with the most effective inhibition of AIMP2 aggregation bind to AIMP2, disaggregate the pre-formed AIMP2 aggregates, and prevented AIMP2/α-synuclein coaggregation and cytotoxicity in SH-SY5Y cells. Moreover, AIMP2 inhibitors prevented α-synuclein preformed fibril (PFF)-induced pathological AIMP2 aggregation in both mouse cortical and embryonic stem cell-derived human dopaminergic neurons, thereby blocking PFF-induced α-synuclein aggregation and neurotoxicity. Collectively, our results suggest that the use of brain-permeable AIMP2 aggregation inhibitors may serve as an effective therapeutic strategy for α-synucleinopathy in PD.

Separation of N'-nitrosonornicotine isomers and enantiomers by supercritical fluid chromatography tandem mass spectrometry.

N'-nitrosonornicotine (NNN) is one of the most prevalent and toxic tobacco-specific nitrosoamines. A chiral center at its 2'-position results in R and S enantiomers, the partial double bond character of the NN = O group also results in E and Z isomers, therefore, NNN can form a total of four absolute configurations (E-(R)-NNN, E-(S)-NNN, Z-(R)-NNN, and Z-(S)-NNN). This study investigated the resolution of R/S enantiomers and E/Z isomers of NNN by supercritical fluid chromatography tandem mass spectrometry (SFC-MS/MS). The baseline separation of E/Z-(R,S)-NNN isomers/enantiomers was accomplished through the optimization of chiral columns and co-solvents. Due to the lack of single standard of E/Z isomers, only R-NNN (sum of E-(R)-NNN and Z-(R)-NNN) and S-NNN (sum of E-(S)-NNN and Z-(S)-NNN) were further examined. Through the comprehensive optimization of SFC-MS/MS conditions, R-NNN and S-NNN were separated with a run time of 5 min, the developed method was validated, and its applicability to the determination of NNN enantiomers in burley tobacco samples was demonstrated. This study could be applied to preparative separation of single enantiomer and/or isomer of NNN, and could provide potential benefits to biologic activity studies on these enantiomers and isomers.

Analysis of waterpipe aerosol constituents in accordance with the ISO standard 22486.

This study analyzed commercial waterpipe tobacco products in accordance with the newly developed ISO 22486 as well as with commercial waterpipes and charcoals using the ISO 22486 puffing regime for comparison. The aerosols from these products were analyzed for their nicotine, humectant, tobacco specific nitrosamine, carbonyl, benzo[a]pyrene, and metal yields. Significant differences were observed among the waterpipe tobacco products when analyzed in accordance with the ISO standard 22486 and with different commercial waterpipes and charcoals. The concentrations of CO and benzo[a]pyrene observed in the consumers' configuration using the ISO 22486 puffing regime (with lit charcoal) were higher than those obtained with the ISO standard using electrical heating, with the yields for carbonyl compounds being lower or higher. The use of the recently published ISO standard for generating water pipe tobacco aerosols should be complemented with analysis by using the consumers' configuration. The necessity for this was demonstrated by the differences in CO and benzo[a]pyrene yields in the present work. It appears that the temperature (280°C) selected for electrical heating of waterpipe tobacco products in ISO 22486 is somewhat lower than that obtained with commercial charcoals, resulting in a generally lower yield of nicotine and total collected matter. In addition, there is a need to evaluate the contribution of commercial charcoals to the concentration of constituents in waterpipe aerosols. This is particularly true for compounds resulting from charcoal combustion, such as CO and benzo[a]pyrene.

Use of autoclave extraction-supercritical fluid chromatography/tandem mass spectrometry to analyze 4-(methylintrosamino)-1-(3-pyridyl)-1-butanone and N'-nitrosonornicotine in tobacco.

4-(methylintrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'-nitrosonornicotine (NNN) are the most prevalent and toxic tobacco specific nitrosamines (TSNAs). Due to their carcinogenicity, knowledge of the composition of NNK and NNN in tobacco is necessary. Herein, a sensitive and rapid method, which employs autoclave extraction-supercritical fluid chromatography/tandem mass spectrometry (SFC-MS/MS), has been developed for the analysis of NNK and NNN in tobacco. Both water-soluble and matrix-bound NNK and NNN were extracted with 100 mM ammonium acetate in an autoclave (130 °C, 4 h), and the aqueous extract was subjected to solvent replacement prior to SFC-MS/MS analysis. NNK and NNN were effectively separated within 5 min by using supercritical CO2 as the main mobile phase coupled with a co-solvent of methanol. Excellent linearity was obtained with coefficients of determination (R2) greater than 0.9997 in the range of 1-160 ng/mL and 5-800 ng/mL for NNK and NNN, respectively. The recoveries were in the range of 92.5-110.0% at different spiked levels of real samples. 12 tobacco samples which include 3 typical tobacco varieties of burley, flue-cured, and oriental tobaccos had been analyzed, and the fraction of matrix-bound NNK was determined as well. In addition, a comparison between the proposed SFC-MS/MS method and a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) internal standard method was conducted. Both techniques exhibit comparable analysis results, but peak splitting of NNN was observed by LC-MSMS due to the existence of E/Z isomers, while SFC-MS/MS offers great improvement through elution condition optimization, demonstrating the applicability of SFC-MS/MS as an alternative tool for NNK and NNN analysis.

Metabolic Activation and Carcinogenesis of Tobacco-Specific Nitrosamine N'-Nitrosonornicotine (NNN): A Density Function Theory and Molecular Docking Study.

N'-nitrosonornicotine (NNN) is one of the tobacco-specific nitrosamines (TSNAs) that exists widely in smoke and smokeless tobacco products. NNN can induce tumors in various laboratory animal models and has been identified by International Agency for Research on Cancer (IARC) as a human carcinogen. Metabolic activation of NNN is primarily initiated by cytochrome P450 enzymes (CYP450s) via 2'-hydroxylation or 5'-hydroxylation. Subsequently, the hydroxylating intermediates undergo spontaneous decomposition to generate diazohydroxides, which can be further converted to alkyldiazonium ions, followed by attacking DNA to form various DNA damages, such as pyridyloxobutyl (POB)-DNA adducts and pyridyl-N-pyrrolidinyl (py-py)-DNA adducts. If not repaired correctly, these lesions would lead to tumor formation. In the present study, we performed density functional theory (DFT) computations and molecular docking studies to understand the mechanism of metabolic activation and carcinogenesis of NNN. DFT calculations were performed to explore the 2'- or 5'- hydroxylation reaction of (R)-NNN and (S)-NNN. The results indicated that NNN catalyzed by the ferric porphyrin (Compound I, Cpd I) at the active center of CYP450 included two steps, hydrogen abstraction and rebound reactions. The free energy barriers of the 2'- and 5'-hydroxylation of NNN are 9.82/8.44 kcal/mol (R/S) and 7.99/9.19 kcal/mol (R/S), respectively, suggesting that the 2'-(S) and 5'-(R) pathways have a slight advantage. The free energy barriers of the decomposition occurred at the 2'-position and 5'-position of NNN are 18.04/18.02 kcal/mol (R/S) and 18.33/19.53 kcal/mol (R/S), respectively. Moreover, we calculated the alkylation reactions occurred at ten DNA base sites induced by the 2'-hydroxylation product of NNN, generating the free energy barriers ranging from 0.86 to 4.72 kcal/mol, which indicated that these reactions occurred easily. The docking study showed that (S)-NNN had better affinity with CYP450s than that of (R)-NNN, which was consistent with the experimental results. Overall, the combined results of the DFT calculations and the docking obtained in this study provide an insight into the understanding of the carcinogenesis of NNN and other TSNAs.

Video-based kinetic analysis of calcification in live osteogenic human embryonic stem cell cultures reveals the developmentally toxic effect of Snus tobacco extract.

Epidemiological studies suggest tobacco consumption as a probable environmental factor for a variety of congenital anomalies, including low bone mass and increased fracture risk. Despite intensive public health initiatives to publicize the detrimental effects of tobacco use during pregnancy, approximately 10-20% of women in the United States still consume tobacco during pregnancy, some opting for so-called harm-reduction tobacco. These include Snus, a type of orally-consumed yet spit-free chewing tobacco, which is purported to expose users to fewer harmful chemicals. Concerns remain from a developmental health perspective since Snus has not reduced overall health risk to consumers and virtually nothing is known about whether skeletal problems from intrauterine exposure arise in the embryo. Utilizing a newly developed video-based calcification assay we determined that extracts from Snus tobacco hindered calcification of osteoblasts derived from pluripotent stem cells early on in their differentiation. Nicotine, a major component of tobacco products, had no measurable effect in the tested concentration range. However, through the extraction of video data, we determined that the tobacco-specific nitrosamine N'-nitrosonornicotine caused a reduction in calcification with similar kinetics as the complete Snus extract. From measurements of actual nitrosamine concentrations in Snus tobacco extract we furthermore conclude that N'-nitrosonornicotine has the potential to be a major trigger of developmental osteotoxicity caused by Snus tobacco.

Novel approach for selective reduction of NNN in cigarette tobacco filler and mainstream smoke.

Research conducted during past decades to reduce the level of the tobacco specific nitrosamine N-nitrosonornicotine (NNN) and its precursor nornicotine in tobacco yielded identification of three tobacco genes encoding for cytochrome P450 nicotine demethylases converting nicotine to nornicotine. We carried out trials to investigate the effect of using tobaccos containing three non-functional nicotine demethylase genes on the selective reduction of NNN in cigarette tobacco filler and mainstream smoke. Our results indicate that the presence of non-functional alleles of the three genes reduces the level of nornicotine and NNN in Burley tobacco by 70% compared to the level observed in currently available low converter (LC) Burley tobacco varieties. The new technology, named ZYVERT™, does not require a regular screening process, while a yearly selection process is needed to produce LC Burley tobacco seeds for NNN reduction. The reduction of NNN observed in smoke of blended prototype cigarettes is proportional to the inclusion level of tobacco having ZYVERT™ technology. Inclusion of Burley tobacco possessing the new trait into a typical American blend resulted in a selective reduction of NNN in cigarette smoke, while the levels of other Harmful and Potentially Harmful Constituents (HPHC) currently in the abbreviated list provided by the US Food and Drug Administration are statistically equivalent in comparison with the levels obtained in reference prototype cigarettes containing LC Burley.

Analysis of N'-nitrosonornicotine enantiomers in human urine by chiral stationary phase liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry.

We have developed a chiral stationary phase liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry (LC-NSI-HRMS/MS) method to investigate the enantiomeric composition of low parts per trillion amounts of the carcinogen N'-nitrosonornicotine (NNN) in the urine of cigarette smokers and smokeless tobacco users. (S)-NNN is the major enantiomer in tobacco and is more carcinogenic than (R)-NNN in rats, but no data are available on the enantiomeric composition of NNN in humans. The method used [13C6]NNN as an internal standard and [pyridine-D4]nornicotine to monitor possible artifactual formation of NNN, which was found to be less than 2% of the quantified NNN. The enantiomeric composition of NNN (20.5±27.1fmol/mL urine) in 20 cigarette smokers was 67±5% (S)-NNN while that in 10 smokeless tobacco users (67.1±56.7 fmol/mL urine) was 56±3% (S)-NNN. These results demonstrate that the highly carcinogenic (S)-NNN is the major enantiomer in human urine, and that the enantiomeric composition of NNN in human urine is remarkably similar to that in cigarette smoke and smokeless tobacco. This is the first study to combine chiral stationary phase separations with nanoelectrospray ionization and high resolution tandem mass spectrometry to quantify trace levels of enantiomeric metabolites in human urine.

A Survey of N'-Nitrosonornicotine (NNN) and Total Water Content in Select Smokeless Tobacco Products Purchased in the United States in 2015.

This investigation provides an updated survey measuring the levels of N'-nitrosonornicotine (NNN) and water content of a select number of smokeless tobacco products sold in the United States in 2015. A total of 34 smokeless tobacco products were collected and analyzed for NNN and water content using LC-MS/MS and GC-TCD, respectively. Smokeless tobacco products were chosen to obtain a representative sample of the different types of products on the U.S. market. These smokeless products represent 12 of the 25 top-selling smokeless tobacco products according to 2013 Nielsen net sales data while five of the smokeless tobacco products are of lower selling smokeless tobacco products. The NNN levels and the water content of the smokeless tobacco products were determined and compared to previous studies. Although the range of NNN levels found was broad for the examined smokeless tobacco products (0.64-12.0 µg/g dry weight), dry snuff had the highest levels of NNN observed (>5 µg/g dry weight). We observed a general decrease in NNN levels for the same six moist snuff products that were analyzed in 2004 compared to our current 2015 study. The water content of the smokeless tobacco products surveyed ranged from 3.92 to 54.8%.

Transcriptome profiling in oral cavity and esophagus tissues from (S)-N'-nitrosonornicotine-treated rats reveals candidate genes involved in human oral cavity and esophageal carcinogenesis.

Recently, we have shown that (S)-N'-Nitrosonornicotine [(S)-NNN], the major form of NNN in tobacco products, is a potent oral cavity and esophageal carcinogen in rats. To determine the early molecular alterations induced by (S)-NNN in the oral and esophageal mucosa, we administered the carcinogen to rats in the drinking water for 10 wk and global gene expression alterations were analyzed by RNA sequencing. At a false discovery rate P-value < 0.05 and fold-change ≥2, we found alterations in the level of 39 genes in the oral cavity and 69 genes in the esophagus. Validation of RNA sequencing results by qRT-PCR assays revealed a high cross-platform concordance. The most significant impact of exposure to (S)-NNN was alteration of genes involved in immune regulation (Aire, Ctla4, and CD80), inflammation (Ephx2 and Inpp5d) and cancer (Cdkn2a, Dhh, Fetub B, Inpp5d, Ly6E, Nr1d1, and Wnt6). Consistent with the findings in rat tissues, most of the genes were deregulated, albeit to different degrees, in immortalized oral keratinocytes treated with (S)-NNN and in non-treated premalignant oral cells and malignant oral and head and neck squamous cells. Furthermore, interrogation of TCGA data sets showed that genes deregulated by (S)-NNN in rat tissues (Fetub, Ly6e, Nr1d1, Cacna1c, Cd80, and Dgkg) are also altered in esophageal and head and neck tumors. Overall, our findings provide novel insights into early molecular changes induced by (S)-NNN and, therefore, could contribute to the development of biomarkers for the early detection and prevention of (S)-NNN-associated oral and esophageal cancers. © 2016 Wiley Periodicals, Inc.

Combined analysis of N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in the urine of cigarette smokers and e-cigarette users.

A liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI(+)-MS/MS) method for the analysis of the tobacco-specific carcinogens N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and their glucuronides (total NNN and total NNAL) in human urine was developed. The method has excellent accuracy and intra-day and inter-day precision, and limits of quantitation of 0.015 and 0.075pmol/mL urine, respectively, for total NNN and total NNAL. A unique aspect of this method is internal assessment of possible artifactual formation of NNN by inclusion of the monitor amine [pyridine-D4]nornicotine. We found that artifactual formation of NNN comprised only 2.5% of the measured amounts of total NNN in urine of cigarette smokers, under our conditions using ammonium sulfamate as an inhibitor of nitrosation. The method was applied to urine samples from cigarette smokers and e-cigarette users. Levels of total NNN and total NNAL in the urine of cigarette smokers averaged 0.060±0.035pmol/mL and 2.41±1.41pmol/mL urine, (N=38), respectively, which were both significantly greater than in the urine of 27 e-cigarette users.

Tobacco-specific N-nitrosamine exposures and cancer risk in the Shanghai Cohort Study: remarkable coherence with rat tumor sites.

The tobacco-specific nitrosamines N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are potent carcinogens for the rat esophagus and lung, respectively. Consistent with the animal carcinogenicity data, we previously reported a remarkably strong association between prospectively measured urinary total NNN, a biomarker of human NNN intake, and the risk of developing esophageal cancer among smokers in the Shanghai Cohort Study. We also demonstrated that urinary total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), a biomarker of exposure to the lung carcinogen NNK, is strongly associated with the risk of lung, but not esophageal cancer in smokers. In this study, we investigated the potential relationship between NNN intake and lung cancer risk in the same cohort. The prospectively collected urine samples from lung cancer cases and matching controls selected for this study, all current smokers, have been previously analyzed for total NNAL, cotinine (a biomarker of nicotine intake) and phenanthrene tetraol (PheT) (a biomarker of exposure to polycyclic aromatic hydrocarbons). Urinary levels of total NNN were not associated with the risk of lung cancer: odds ratios (95% confidence intervals) associated with the second and third tertiles of total NNN, relative to the lowest tertile, were 0.82 (0.36-1.88) and 1.02 (0.39-2.89), respectively (p for trend = 0.959), after adjustment for self-reported smoking history, urinary cotinine and PheT. The results of this study reaffirm the previously reported specificity of urinary total NNN and total NNAL as predictors of esophageal and lung cancer risks, respectively, in smokers, and demonstrate remarkable coherence between rat target tissues of these carcinogens and susceptibility to cancer in smokers.

Estimation of mouth level exposure to smoke constituents of cigarettes with different tar levels using filter analysis.

A nicotine part-filter method can be applied to estimate smokers' mouth level exposure (MLE) to smoke constituents. The objectives of this study were (1) to generate calibration curves for 47 smoke constituents, (2) to estimate MLE to selected smoke constituents using Japanese smokers of commercially available cigarettes covering a wide range of International Organization for Standardization tar yields (1-21mg/cigarette), and (3) to investigate relationships between MLE estimates and various machine-smoking yields. Five cigarette brands were machine-smoked under 7 different smoking regimes and smoke constituents and nicotine content in part-filters were measured. Calibration curves were then generated. Spent cigarette filters were collected from a target of 50 smokers for each of the 15 brands and a total of 780 filters were obtained. Nicotine content in part-filters was then measured and MLE to each smoke constituent was estimated. Strong correlations were identified between nicotine content in part-filters and 41 out of the 47 smoke constituent yields. Estimates of MLE to acetaldehyde, acrolein, 1,3-butadiene, benzene, benzo[a]pyrene, carbon monoxide, and tar showed significant negative correlations with corresponding constituent yields per mg nicotine under the Health Canada Intense smoking regime, whereas significant positive correlations were observed for N-nitrosonornicotine and (4-methylnitrosoamino)-1-(3-pyridyl)-1-butanone.

Molecular genetics of alkaloid biosynthesis in Nicotiana tabacum.

Alkaloids represent an extensive group of nitrogen-containing secondary metabolites that are widely distributed throughout the plant kingdom. The pyridine alkaloids of tobacco (Nicotiana tabacum L.) have been the subject of particularly intensive investigation, driven largely due to the widespread use of tobacco products by society and the role that nicotine (16) (see Fig. 1) plays as the primary compound responsible for making the consumption of these products both pleasurable and addictive. In a typical commercial tobacco plant, nicotine (16) comprises about 90% of the total alkaloid pool, with the alkaloids nornicotine (17) (a demethylated derivative of nicotine), anatabine (15) and anabasine (5) making up most of the remainder. Advances in molecular biology have led to the characterization of the majority of the genes encoding the enzymes directly responsible the biosynthesis of nicotine (16) and nornicotine (17), while notable gaps remain within the anatabine (15) and anabasine (5) biosynthetic pathways. Several of the genes involved in the transcriptional regulation and transport of nicotine (16) have also been elucidated. Investigations of the molecular genetics of tobacco alkaloids have not only provided plant biologists with insights into the mechanisms underlying the synthesis and accumulation of this important class of plant alkaloids, they have also yielded tools and strategies for modifying the tobacco alkaloid composition in a manner that can result in changing the levels of nicotine (16) within the leaf, or reducing the levels of a potent carcinogenic tobacco-specific nitrosamine (TSNA). This review summarizes recent advances in our understanding of the molecular genetics of alkaloid biosynthesis in tobacco, and discusses the potential for applying information accrued from these studies toward efforts designed to help mitigate some of the negative health consequences associated with the use of tobacco products.